DEVELOPMENT OF A SYNTHETIC PEPTIDE VACCINE AGAINST H5N1 BASED ON MHC-I EPITOPES بحث في تطوير لقاح الببتيد المصنع ضد فيروس إنفلونزا الطيور المستند على

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1 DEVELOPMENT OF A SYNTHETIC PEPTIDE VACCINE AGAINST H5N1 BASED ON MHC-I EPITOPES بحث في تطوير لقاح الببتيد المصنع ضد فيروس إنفلونزا الطيور المستند على حواتم أليالت المركب الرئيسي للتالؤم النسيجي.) MHC-I ( Step 2: verification of the peptide candidate FLKDVMESM through ELISA and IFN-γ ELISPOT analysis in a laboratory setting Noha Abdulwahab, Samir Mourad, Samah R. Borghol Institute for Genetic Engineering, Ecology and Health (IGEEH) Karlsruhe, Germany Postal Address: Verein für Gentechnik, Ökologie und Gesundheit (VGÖG) e.v., Haid-und-Neu-Str.7, Karlsruhe, Germany مركز ابحاث الشرق االوسط للجينات والتقنية البيولوجية رأسنحاش قضاء الب تون - لبنان Middle East Genetics and Biotechnology Institute (MEGBI) Main Road, Ras Nhache, Batroun, Lebanon, info@aecenar.com

2 Last update: 2-Jun-11 Contents 4...نظرة عامة / Overview 5...المقدمة / Introduction النظرية االساسية / basis 2 Theoretical 2.1 Influenza-A-virus H5N1 / " يآ " للفريوس H5N1 انفلونزا... 7 ىيكل جزيئات الفريوس وجينوم فريوس / virus Structure of virus particles and the genome of influenza A 7...االنفلونزا آي 8...ىيكل اجلينوم لفريوس االنفلونزا / virus Genome structure of influenza 13...مقعد التوافق النسيجي الكبري اجلزئي / Molecule 2.2 MHC-I بيتا 2 ميك روغلوبيلني β2m./ MATERIAL AND METHOD A- Part I Cloning of the HLA-A*0201 and b-2m gene from PBMC Expression of HLA-A*0201 and b-2m Prepare pet Vector: Prepare the insert DNA with the adapter LIGATION Transformation INTO EXPRESSION HOST Extraction and solubilization of inclusion bodies ELISA assay of peptide-mhc complex formation: More detials: B- Part II

3 3.6 Amplification of the HLA-A*0201 and β-2m gene from PBMC Prepare the insert DNA (HLA-A*0201 and β2m) with the adapter Expression of HLA-A*0201 and β-2m Vector preparation: Ligation Transformation Extraction and solubilization of inclusion bodies ELISA assay of peptide-mhc complex formation: For more details: النتائج / Results 4 مضاعفة جني ىال* 0204 ويبتا- 2 آم من احمليط اخلارجي / PBMC 4.4 Amplification of the HLA-A*0204 and β-2m gene from 51...خلاليا الدم البيضاء ربضري الناقل / vector 4.2 Preparation of 57...عملية الربط / Ligation عملية النقل / Transformation Appendix: References

4 و. نظرة عامة / Overview The research of development of a synthetic peptide vaccine against H5N1 based on MHC-I epitopes require four steps. The step 2 is the step described in detail in this document, which is a qualitative ELISA test. By this test we have verified the actual binding affinity between the synthetically peptide KLDVMESM which has been predicted by NetCTL 1.2 in the first step of the research and the HLA- A*0201 which have been extracted and purified from the white blood cells and the measured qualitative affinity was the yellow color of this complex that we have seen it in the last result of ELISA test. This document contains in the first section the concepts and scientific terms that used in the research, the second section includes protocols that are used for the application of this stage is taken from the Internet sites, The third section contains the same protocols that in the second section with some modifications that commensurate with the devices and materials that presented in the laboratory, the fourth section presents the recent results with images of the experiments that have been working in the MEGBI laboratory. Finally, the time which took for each stage of this process is added in the last section of this document إن حبث تطوير لقاح الببتيد ادلصنع ضد فريوس إنفلونزا الطيور ادلستند على حوامت النسيجي.) MHC-I ( ادلرحلة الثانية إختبار إيليزا النوعي أليالت ادلركب الرئيسي للتالؤم يستوجب أربع مراحل إلسبامو منها ادلشروحة بالتفصيل يف ىذه الوثيقة الذي حقق لنا الببتيد ادلصنع KLDVMESM األوىل بواسطة واليت ىي نسبة الربط الفعلي بني الذي مت تنبؤه يف ادلرحلة اذلال NetCTL 1.2 اليت مت إستخراجها وتنقيتها من كريات الدم البيضاء وادلقياس النظري حلقيقة ىذا الربط ىو اللون األصفر الذي رأيناه يف التنيجة األخرية لفحص إيليزا. تتضمن ىذه الوثيقة يف القسم األول منو ادلفاىيم وادلصطلحات العلمية ادلستخدمة يف البحث القسم الثاين يتضمن الربوتوكوالت ادلستخدمة لتطبيق ىذه ادلرحلة وىي مأخوذة من مواقع عن اإلن تنت أما القسم الثالث يتضمن نفس بروتوكوالت القسم الثاين مع إجراء بعض التعديالت عليها دبا يتناسب مع اآلالت وادلواد ادلوجودة يف ادلخترب أما القسم الرابع يعرض النتائج األخرية مع صور من التجارب اليت مت العمل عليها يف سلترب MEGBI اجلدول إلسبامها. وأخريا مضاف إىل ىذه الو اجلدوثيقة الزمين اليت إستغرقتو كل عملية من ىذه ادلرحلة 4

5 المقدمة / Introduction 1 Avian influenza is caused by the influenza-a-a virus H5N1 which is found in birds. The influenza- A-type virus is characterized by the HA (hemagluttinin) and NA (neuraminidase) proteins. H5N1 has one HA type 5 proteins (H5) as well as one NA type 1 protein (N1). The majority of humans do not possess any kind of immunity against the H5N1 virus. The virus is able to replicate efficiently inside the human body, for this reason should be opposite this pandemic. However, a mutation in the H5N1 virus, only two amino acid exchanges at the HA receptor binding site of H5N1 are required in order to optimize binding of the virus to N-acetyl-neuraminicacid, which is found on epithelial cells of the human lung. It causes a fast and effective spreading of H5N1 in the human population. And the goal of the research is the identification of peptides of the potential H5N1 mutants which might elicit an immune response in humans. Humans could be immunized with these peptides before the outbreak of a pandemic. Approach Computer-based analysis of candidate peptides of the mutated H5N1 which bind to MHC-I with high affinity and hence are immunogenic. Verification of these candidates through ELISA and IFN-γ ELISPOT analysis in a laboratory setting. Animal testing of the candidate peptides which have found to be promising in the above mentioned tests. إن فريوسات إنفلونزا آي (H5N1( تسبب إنفلونزا الطيور. ودييز ىذه الفريوسات النوع اخلامس من الربوتيينات السكرية الراصة الدموية hemaglutinin( H5( والنوع األول من بروتيني النورواميديناز )N1 )Neuraminidase ادلغلفة للمادة النووية للفريوس. ودبا إن غالبية البشر ال سبلك أي نوع من احلصانة ضد فريوس H5N1 فإن ىذا الفريوس قادر على التكرار بكفاءة داخل جسم اإلنسان لذا جيب مواجهة ىذا الوباء. إال أن التغيري اجليين ادلفاجئ يف الفريوس من خالل تبادل األضباض األمينية يف مستقبل موقع ملزم لفريوس h5n1 من أجل ربسني ربط الفريوس حبمض أن-أسيتيل نورامينيك الذي يوجد على اخلاليا الظاىرية لرئة اإلنسان يسبب اإلنتشار السريع والفعال لو بني البشر. لتطوير ىذا اللقاح يجب معرفة ببتيدات طفرات H5N1 المحتملة التي قد تثير رد فعل مناعة لدى البشر. المنهج: يستند احلاسوب على ربليل الببتيد ادلرشح للتغري اجليين ادلفاجئ يف فريوس H5N1 الذي يرتبط ب MHC-I يف نسبة عالية لذا فهي ادلناعة. التحقق من ىذه ادلرشاحات من خالل فحص إيليزا و IFN-Y ELISPOT يف ادلخترب. إختبار الببتيدات ادلرشحة اليت وجدت يف الفحوصات ادلذكورة أعاله لتكون زلفزة للمناعة على احليوان. المراحل: ادلرحلة األوىل: ىي إستناد احلاسوب على تنبؤ MHC-I مت إصلازىا يف أدلانيا. ادلرحلة الثانية: ىي إختبار إيليزا الكمي للتحقق من نسبة الربط 5

6 Steps: Step 1 Computer-based MHC-I prediction (worked in Germany). Step 2 quantitative ELISA Experiment for investigated from the actual binding affinity between the synthetically peptides and the MHC-I alleles from the respective HLA types for which peptides have been predicted by NetCTL1.2. Step 3 Animal studies and IFN-γ ELISPOT analysis. Step 4 Clinical Studies The second step is the step described in this book and it is now under discussion الفعلي بني الببتيدات ادلصنعة وأليالت ادلركب الرئيسي للتالؤم النسيجي )MHC-I( من مستضدات كريات البيض البشرية NetCTL للببتيدات اليت مت تنبؤىا بواسطة )HLA( IFN-Y ىي الدراسات احليوانية وربليل.1.2 ادلرحلة الثالثة: ELISPOT للتحقق من فعالية لقاح الببتيد. ادلرحلة الرابعة: ىي الدراسات السريرية المرحلة الثانية ىي المرحلة الوارد ذكرىا في ىذا الكتاب وىي اآلن قيد البحث 6

7 النظرية االساسية / basis 2 Theoretical 2.1 Influenza-A-virus H5N1 / انفلونزا "آي" للفيروس H5N1 The avian influenza is causes by the influenza A-virus H5N1 that belongs to the family of Orthomyxoviruses االنفلونزا " يآ " H5N1 ىي اليت تسبب انفلونزا الطيور الذي ينتمي إىل عائلة Orthomyxoviruses ىيكل جزيئات الفيروس وجينوم فيروس االنفلونزا / virus Structure of virus particles and the genome of influenza A آي The influenza virus consists of 8 negative-strand RNA molecules surrounded by an envelope. The envelope contains the HA and NA proteins. Influenza enters cells by receptormediated endocytosis Once inside the host, the viral RNAs are transcribed in the nucleus, stealing 5' caps from host mrnas. These are then translated at ribosomes. i 1 فريوس احلمض االنفلونزا الرييب النووي من يتكون السلبية سالالت 8 زلاطة دبغلف. ادلغلف حيتوي على الربوتينات ىا.NA و نا HA األنفلونزا يدخل بوساطة.مستقبالت االلتقام. اخلاليا ينسخ احلمض النووي الرييب الفريوسي يف النواة مرة واحدة داخل اخللية ادلضيفة ويأخذ قبعة 5' من احلمض النووي الرييب ادلضيف. مث يتم ربويلها اىل ريبوسومات 1 7

8 On the outside surface of the influenza virus is a lipid bilayer with HA, NA and M2 proteins inserted into it. Inside the bilayer are eight separate, linear RNA segments that make up the viral genome ii2. السطح اخلارجي لفريوس االنفلونزا ىو طبقة ثنائية من الدىون مع HA و NA و الربوتينات M2 ادلدرجة فيو. داخل ىذه الطبقة الثنائية شرائح احلمض النووي الرييب الثمانية اليت ىتشكل اجلينوم الفريوسي. ىيكل الجينوم لفيروس االنفلونزا / virus Genome structure of influenza Segment: Size(nt) Polypeptide(s) Function PB2 Transcriptase: cap binding PB1 Transcriptase: elongation PA Transcriptase: protease activity (?) HA Haemagglutinin NP Nucleoprotein: RNA binding; part of transcriptase complex; nuclear/cytoplasmic transport of vrna NA Neuraminidase: release of virus M1 M2 NS1 NS2 Matrix protein: major component of virion Integral membrane protein - ion channel Non-structural: nucleus; effects on cellular RNA transport, splicing, translation. Anti-interferon protein. Non-structural: nucleus+cytoplasm, function unknown Genetics of influenza viruses. Ann Rev Genet :

9 A representation of the flu virus that shows the outer shell and a cutaway revealing the 8 RNA pieces that comprise the genome of the virus. The outer shell is composed of lipids obtained from the last host cell. This is decorated with hemagglutinin (HA, yellow) and neuraminidase (NA, pink). HA is necessary for entrance into cells, while NA is needed for release from cells. NP is the viral polymerase. ىذا رسم الغالف لفريوس اخلارجي االنفلونزا ويكشف الذي عن يظهر 8 قطع من احلمض اجلينوم النووي للفريوس. يشكل اليت الرييب وتتألف القشرة اخلارجية من الدىون اليت مت احلصول عليها من اخللية ادلضيفة ادلاضية. ىذا مع ىيماغلوتينني والنورامينيداز ضروري أن للدخول )نا وردي(. إىل ويزين )ىا أصفر( ىا ىو اخلاليا يف حني يساعد على اخلروج منها. )NP( NA النيكليوبروتني الفريوس. ىو بوليمرياز These copies the RNA genome into mrna for protein synthesis and later in the life cycle makes copies of each RNA for new viral particles. NP is very error prone and creates many mutations in the viral genome. HA and NA therefore change rapidly, and escape recognition by our immune systems. The genome of the virus is composed of eight (-) single-stranded RNAs (these (-) strands cannot be translated into protein) with each segment being complementary to one mrna. Six of the eight mrnas ىذه النسخ من الرنا اجلينومي داخل الرنا "م" لتصنيع الربوتني وبعد ذلك يف دورة احلياة جيعل نسخ لكل ضبض نووي رييب جلزيئات فريوسية جديدة. نيكليوبروتني ىو معرض جدا للخطأ وخيلق العديد من الطفرات يف اجلينوم الفريوسي وبالتايل الطفرات. "ىا" و "نا" تتغري بسرعة لتهرب قبل أن تتعرف عليها أنظمة ادلناعة لدينا. ويتكون اجلينوم للفريوس من شبانية سالالت سلبية للرنا ادلفرد )ىذه السالالت السلبية ال ديكن أن ت تجم إىل فروع الربوتني( مع كل قطعة يتم اكتمال mrna واحد. ستة من شبانية mrna ترمز لنوع واحد من الربوتينات يف حني أن اإلثنان ادلتبقيان يرمزان إىل إثنني من الربوتينات بواسطة 9

10 code for single proteins, while the remaining two code for two proteins by differential splicing of the RNA. Each mrna segment is associated with multiple copies of the nucleocapsid protein (NP) and an RNA polymerase (made from the viral proteins PB1, PB2 and PA) 3. الربط التفضلي من احلمض النووي الرييب. ويرتبطكل جزء من mrna مع نسخ متعددة من النيكلويوبروتني وبوليمرياز احلمض النووي الرييب )مصنوعة من بروتينات فريوسية PB2 PB1 وPA (.. iii4 1 الا. لتااق 1. Adsorption The virus becomes attached to the cells, and at this stage, it can be recovered in the infectious form without cell lysis by procedures that either destroy the receptors or weaken their bonds to the virions. Animal viruses have specialized attachment sites distributed over the surface of the virion e.g. orthomyxoviruses and paramyxoviruses attach through glycoprotein يف ىذه ادلرحلة تلتصق الفريوس على اخللية و كدي نها إستعادة عافيتها على الشكل ادلعدي دون ربلل اخللية بواسطة االجراءات اليت تدمر ادلستقبالت أو تضعف روابطهم اىل االجسام الفريوسية. الفريوسات احليوانية سبتلك مواقع متخصصة لاللتصاق موزعة على سطح اجلسم الفريوسي.مثل paramyxoviruses و orthomyxoviruses يلتصقان من خالل اجلليكوبروتني و Source: 10

11 ال. spikes, and adenoviruses attach through the penton fibers. Adsorption occurs to specific cellular receptors. Some receptors are glycoproteins, others are phospholipids or glycolipids. These are usually macromolecules with specific physiological functions, such as complement receptors for EBV. Whether or not receptors for a certain virus are present on a cell depends on the species, the tissue and its physiological state. Cells lacking specific receptors are resistant. Attachment is blocked by antibodies that bind to the viral or cellular sites involved. بلتصق من خالل اخلماسية. االلياف االلتصاق adenoviruses حيدث دلستقبالت خاليا معينة و االخرين ىم ضخمة ضمن الفوسفوليبيد او فيزيولوجية وظائف بعض ادلستقبالت ىي اجلليكوليبيد. اجلليكوبروتني وعادة تكون جزيئات زلددة مثل تكملة دلستقبالت.EBV سواء كانت أم مل تكن مستقبالت لفريوسات معينة موجودة على خلية تعتمد على األنواع واألنسجة وحالتو الفيزيولوجية. تفتقر اخلاليا اىل مستقبالت خاصة مقاومة. مت منع التعلق عن طريق ادلضادات احليوية اليت تربط ادلواقع ادلش تكة بني الفريوس او اخلاليا. 2. Penetration 2 دخول Penetration rapidly follows adsorption, and the virus can no longer be recovered from the intact cell. The most common mechanism is receptor mediated endocytosis, the process by which many hormones and toxins enter cells The virion is endocytosed and contained within a cytoplasmic vacuole الدخول السريع يلي عملية االمتصاص وىنا ال يستطيع الفريوس من اس تداد عافيتو من خاليا سليمة. االلتقام بواسطة ادلستقبالت ىو االلية االكثر شيوعيا. حيث من خالذلا يدخل العديد من اذلرمونات و السموم إىل اخلاليا. تلتقم أجزاء الفريوس داخل ذبويف سيتوبالمسي يف اخللية 3.نزع الغالف 3. Uncoating A key step in uncoating is the acidification of the content of the endosome to a ph of about 5, owing to the activity of a proton pump present in the membrane The low ph causes rearrangement of coat components, which then expose normally اخلطوة االساسية يف نزع الغالف ىي يف نسبة األسيد ادلوجود) PH 5( يف الدخلول )ىو حيز زلاط بالغشاء اخللوي )endosome وذلك بسبب مضخة الربوتني ادلوجودة يف الغالف. يسبب اخنفاض الرقم اذليدروجيين إىل إعادة مكونات الطبقة,الذي يكشف عادة ادلواقع الغري ادلائية اليت تربط ادلادة الدىنية يف الطبقة 11

12 hidden hydrophobic sites. They bind to the lipid bilayer of the membrane, causing the extrusion of the viral core into the cytosol. For influenza virus, the acid-sensitive component is the core HA2 unit of the haemagglutinin, for adenoviruses, it is the penton base. الثنائية للغشاء شلا تسبب يف قدف النواة الفريوسي داخل السيتوسول. عنصر األضباض احلساسة ىي وحدة ال HA 2 األساسية من اذليماغلوتينني لفريوس االنفلونزا وىي القاعدة اخلماسية لل adenoviruses iv5 النسخ المتماثل للحمض النووي الفيروسي.4 Viral Nucleic Acid Replication Virulent viruses, either DNA and RNA, shut off cellular protein synthesis and disaggregate cellular polyribosomes, favouring a shift to viral synthesis The mechanism of protein synthesis shut-off varies even within the same viral family Poliovirus, using a viral protease, causes cleavage الفريوسات الفتاكة سواء الرنا أو الدنا توقف تصنيع بروتينات اخللية و تفصل البولرييبوسوم لصاحل حدوث ربول يف تصنيع الفريوس. إن إيقاف آلية تصنيع الربوتني زبتلف حىت داخل األسرة الفريوسية نفسها فريوس شلل األطفال يستخدم انزمي لقطع الربوتني الفريوسي 5 Paul Digard, Dept Pathology, University of Cambridge 12

13 of a 200 Kd cap-binding protein, which is required for initiation of translation of capped cellular messengers. In contrast to virulent viruses, moderate viruses e.g. polyomaviruses may stimulate the synthesis of host DNA, mrna, and protein This phenomenon is of considerable interest for viral carcinogenesis. Maturation and Release Maturation proceeds differently for naked, enveloped, and complex viruses v 6 ويسبب االنقسام ل 200 Kd لقبعة-ادللزمة للربوتينات وىو مطلوب للبدء يف ترصبة رسائل اخلاليا ادلغطاة. وعلى العكس من الفريوسات الفتاكة ىناك فريوسات معدلة مثل polyomaviruse ديكنها أن ربفز تصنيع الرنا"م" والدنا و الربوتينات للخلية ادلضافة ىذه الظاىرة أخذت قدراكبريا من اإلىتمام يف السرطان الفريوسي. النضوج و التحرر نضوج احلصيلة بطريقة سلتلفة عن الفريوسات اجملردة ادلغلفة و ادلعقدة مقعد التوافق النسيجي الكبير الجزئي / Molecule 2.2 MHC-I MHC molecules are membrane-bound proteins: MHC I molecules are found on almost all tissues of the body, while MHC II molecules are found only on antigenpresenting cells. MHC molecules possess a deep groove that is capable of holding a short peptide. MHC I molecules process proteins present inside the cell and present them on their surface. MHC II molecules present antigens taken from the phagosome digestion, most often foreign cells, and present them to the immune system. جزيئات معقد التوافق النسيجي الكبري ىي بروتينات غشاء زلددة: مت العثور على جزيئات معقد التوافق النسيجي الكبري األول يف أنسجة كلها تقريبا من اجلسم يف حني مت العثور على جزيئات معقد التوافق النسيجي الكبري الثاين فقط على مستضد تقدمي اخلاليا. جزيئات معقد التوافق النسيجي الكبري سبتلك ذبويف عميق قادر على عقد الببتيد القصري. عملية جزيئات معقد التوافق النسيجي الكبري األول معاجلة الربوتينات ادلوجودة داخل اخللية و عرضها على سطحها. أما جزيئات معقد التوافق النسيجي الكبري الثاين يظهر ادلستضدات ادلأخوذة من خاليا اذلضم ويف أغلب األحيان اخلاليا األجنبية وعرضها على اجلهاز ادلناعي. The immune system monitors the proteins present on MHC I molecules and activates when a foreign protein, from an intracellular اجلهاز ادلناعي يراقب الربوتينات ادلوجودة على جزيئات معقد التوافق النسيجي الكبري األول وينشط عندما يتم الكشف عن الربوتينات

14 parasite, is detected. This normally results in the destruction of the cell. األجنبية من الطفيليات داخل اخلاليا. ىذه النتائج تظهر عادة يف تدمري اخللية Structure of an MHC molecule بيتا 2 ميكروغلوبيلين β2m./ 2.3 This gene encodes a serum protein found in association with the major histocompatibility complex (MHC) class I heavy chain on the surface of nearly all nucleated cells. The protein has a predominantly beta-pleated sheet structure that can form amyloid fibrils in some pathological conditions. A mutation in this gene has been shown to result in hypercatabolic hypoproteinemia vi7 يرمز ىذا اجلني على بروتني ادلصل ادلوجود بالتعاون مع معقد التوافق النسيجي الكبري الصنف األول للسلسلة الثقيلة على سطح ما يقارب صبيع اخلاليا اليت سبتلك نواة. الربوتني لو ىيكل ورقة مطوية-بيتا يف الغالب اليت ديكن أن تشكل ألياف نسيجية أميلودية يف بعض احلاالت ادلرضية. وقد تبني طفرة يف ىذا اجلني إىل نقص بروتني الدم ادلفرط األيض )hypercatabolism(

15 3 MATERIAL AND METHOD A- Part I The sequence of HLA-A*0201 was extracted from ncbi: ORIGIN 1 atggccgtca tggcgccccg aaccctcgtc ctgctactct cgggggctct ggccctgacc 61 cagacctggg cgggctctca ctccatgagg tatttcttca catccgtgtc ccggcccggc 121 cgcggggagc cccgcttcat cgcagtgggc tacgtggacg acacgcagtt cgtgcggttc 181 gacagcgacg ccgcgagcca gaggatggag ccgcgggcgc cgtggataga gcaggagggt 241 ccggagtatt gggacgggga gacacggaaa gtgaaggccc actcacagac tcaccgagtg 301 gacctgggga ccctgcgcgg ctactacaac cagagcgagg ccggttctca caccgtccag 361 aggatgtatg gctgcgacgt ggggtcggac tggcgcttcc tccgcgggta ccaccagtac 421 gcctacgacg gcaaggatta catcgccctg aaagaggacc tgcgctcttg gaccgcggcg 481 gacatggcag ctcagaccac caagcacaag tgggaggcgg cccatgtggc ggagcagttg 541 agagcctacc tggagggcac gtgcgtggag tggctccgca gatacctgga gaacgggaag 601 gagacgctgc agcgcacgga cgcccccaaa acgcatatga ctcaccacgc tgtctctgac 661 catgaagcca ccctgaggtg ctgggccctg agcttctacc ctgcggagat cacactgacc 721 tggcagcggg atggggagga ccagacccag gacacggagc tcgtggagac caggcctgca 781 ggggatggaa ccttccagaa gtgggcggct gtggtggtgc cttctggaca ggagcagaga 841 tacacctgcc atgtgcagca tgagggtttg cccaagcccc tcaccctgag atgggagccg 901 tcttcccagc ccaccatccc catcgtgggc atcattgctg gcctggttct ctttggagct 961 gtgatcactg gagctgtggt cgctgctgtg atgtggagga ggaagagctc agatagaaaa 1021 ggagggagct actctcaggc tgcaagcagt gacagtgccc agggctctga tgtgtctctc 1081 acagcttgta aagtgtga The sequence of β0-m was extracted from ncbi: ORIGIN 1 aatataagtg gaggcgtcgc gctggcgggc attcctgaag ctgacagcat tcgggccgag 61 atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggaggct 121 atccagcgta ctccaaagat tcaggtttac tcacgtcatc cagcagagaa tggaaagtca 181 aatttcctga attgctatgt gtctgggttt catccatccg acattgaagt tgacttactg 241 aagaatggag agagaattga aaaagtggag cattcagact tgtctttcag caaggactgg 301 tctttctatc tcttgtacta cactgaattc acccccactg aaaaagatga gtatgcctgc 361 cgtgtgaacc atgtgacttt gtcacagccc aagatagtta agtgggatcg agacatgtaa 421 gcagcatcat ggaggtttga agatgccgca tttggattgg atgaattcca aattctgctt 481 gcttgctttt taatattgat atgcttatac acttacactt tatgcacaaa atgtagggtt 541 ataataatgt taacatggac atgatcttct ttataattct actttgagtg ctgtctccat 601 gtttgatgta tctgagcagg ttgctccaca ggtagctcta ggagggctgg caacttagag 15

16 661 gtggggagca gagaattctc ttatccaaca tcaacatctt ggtcagattt gaactcttca 721 atctcttgca ctcaaagctt gttaagatag ttaagcgtgc ataagttaac ttccaattta 781 catactctgc ttagaatttg ggggaaaatt tagaaatata attgacagga ttattggaaa 841 tttgttataa tgaatgaaac attttgtcat ataagattca tatttacttc ttatacattt 901 gataaagtaa ggcatggttg tggttaatct ggtttatttt tgttccacaa gttaaataaa 961 tcataaaact tgatgtgtta tctctta 3.1 Cloning of the HLA-A*0201 and b-2m gene from PBMC 8vii Protocol: 1. Isolate 9 the total RNA that was extracted from the peripheric blood monocytes (PBMC) from 10 ml human_s anticoagulative venous blood was dissolved in 50µL ddh2o. RESULT: total RNA only 2. Quantify by ultraviolet or spectrophotometer. Goal: for incertitude that find RNA if the number < Amplify The extracellular fragment of HLA-A*0201 (including the fragment of transmembrane) using total RNA as a template with the forward primer: 5 - CCCTGACCCAGACCTGGGCGG-3 and the reverse primer 3 - AGGGTCGGGTGGTAGGGGTAG-5.by PCR as follows: RESULT: Amplification the DNA segment of HLA-A*0201 only. 4. Then using the PCR-product as a template amplified the just extracellular fragment of HLA-A*0201 with the forward primer 5 -GGCTCCCACTCCATGAGGTAT-3 and the reverse primer 3 -GGGAGTGGGACTCTACCCTCG -5. RESULT: by nested PCR can surely the amplification of DNA segment of HLA-A* Analogously, we constructed b-2m expression vector; however, there were some differences between them. The fragment encoding b-2m was amplified using total RNA as a template with the forward primer 5 -GCATCCAGCGTACTCCAAAGA-3 and the reverse 8 This step was extracted from Protein Expression and Purification 35 (2004) , form science direct 9 The procedures of this step as follows in peqgold kit and for more details please see the part

17 primer 3 -ATTCACCCTAGCTCTGTACCG-5. The PCR protocol was the same as that used in amplification of HLA-A*0201. RESULT: Amplification the DNA segment of β2m only. 6. After purification from an agarose gel. The DNA sequences were verified by sequencing. 3.2 Expression of HLA-A*0201 and b-2m We referred on this step to expression by petvector with BL21 (DE3) (host strain for expression) Prepare pet Vector: To digest and gel-purify the vector 10viii : Reagent: - pet vector - 10X restriction enzyme buffer - EcoR I restriction enzyme - calf intestinal alkaline phosphatase 1. Assemble the following components in a microcentrifuge tube: 3 μg pet vector 3 μl 02X restriction enzyme buffer U EcoR I restriction enzyme (assuming compatible buffer; the total volume of enzyme added should not exceed 10% of the reaction volume to avoid high glycerol concentrations) x μl Nuclease-free water brought to volume 32 μl Total volume 2. Incubate at the appropriate temperature (usually 37 C) for 2 4 h. 3. Run a 3 μl sample together with Perfect DNA Markers on an agarose gel to check the extent of digestion. 4. When digestion is complete, add calf intestinal alkaline phosphatase (Calbiochem Cat. No ) directly to the remainder of the digestion. The enzyme functions in most restriction This step from Novagen. lifeserv.bgu.ac.il/wb/zarivach/.../novagen%20pet%20system%20manual.pdf 17

18 buffers under the conditions described here. It is important to use the correct amount of enzyme; too much can cause unwanted deletions and can be difficult to remove for future steps. Three μg of a typical pet vector (5 kbp) corresponds to about 0 pmol DNA ends when linearized, or about 4 pmol ends if two enzymes were used for digestion. We recommend using 0.05 units of alkaline phosphatase per pmol ends. Dilute the enzyme in water or 50 mm Tris- HCl, ph 9.0 just before use. 5. Incubate at 37 C for 30 min. 6. Add gel sample buffer to the reaction and load the entire sample into a large well ( cm wide) on a 0% agarose gel containing 2.5 μg/ml ethidium bromide. Run the gel far enough to separate the linear plasmid from nicked and supercoiled species. It is useful to run uncut vector DNA in an adjacent lane to help distinguish undigested from linearized plasmid DNA. 7. Visualize the DNA band with a long wave UV light source and cut the band from the gel using a clean razor blade. Avoid over exposure to the light source, which can cause nicks and double strand breaks in the DNA. 8. Recover the DNA from the gel slice. The SpinPrep Gel DNA Kit (Cat. No ) is ideal for this application. Resuspend the final product in a total volume of 32 μl (usually about 52 ng/μl DNA). The DNA can be quantified spectophotometrically or using the PicoGreen kit from Molecular Probes. Assume recoveries in the range of 50% for the ligation step. 9. Store the treated vector at 20 C until use Prepare the insert DNA with the adapter Suggested Conditions for Adapter Addition to DNA 11ix. Reagent: 1. 5X adapter buffer 2. EcoR I(Not I) adapter (1 mg/ml) M DTT 4. T4 DNA ligase (1 U/μl) 5. T4 polynucleotide kinase. 1. Add the following reagents to a microcentrifuge tube on ice. Up to 5 μg blunt-ended DNA - 10 μl 5X adapter buffer (330 mm Tris-HCl (ph 7.6),50 mm MgCl2, 5 mm ATP) - 10 μl EcoR I(Not I) adapter (1 mg/ml) This step was extracted from invitrogen life technologies. 12 EcoR I(Not I) adapter may be used to add EcoR I cohesive ends directly to any blunt-ended DNA fragment. The adapter is formed by annealing two oligonucleotides together forming a phosphorylated, blunt end and a nonphosphorylated EcoR I half-site. Ligation to blunt ended DNA results in EcoR I half-sites on both ends of the DNA. The adapted DNA may be ligated into any EcoR I containing vector. The adapter also contains the 18

19 - 7 μl 0.1 M DTT - distilled water sufficient to bring the volume to 45 μl. 2. Then add 5 μl of T4 DNA ligase (1 U/μl) and mix gently. 3. Incubate the reaction for a minimum of 16 h at 16 C. 4. Heat the reaction at 70 C for 10 min to inactivate the ligase. 5. Place the reaction on ice. The adapted DNA, after adapter removal (step 9) may be ligated directly to any phosphorylated, EcoR I-digested vector. However, if a dephosphorylated EcoR I-digested vector is used, the adapted cdna must be phosphorylated first before adapter removal (step 6). 6. Add 3 μl (30 units) of T4 polynucleotide kinase to the reaction from step Mix gently, and incubate the reaction for 30 min at 37 C. 8. Heat the reaction at 70 C for 10 min to inactivate the kinase and place the reaction on ice. 9. Remove the excess EcoR I(Not I) adapters by column chromatography (ie. cdna Size Fractionation Columns, Cat. No ) or by some other method (ie. gel electrophoresis) and then ligate into the appropriate vector. Reagent: LIGATION - 10X Ligase Buffer (200 mm Tris-HCl ph 7.6, mm MgCl2-100 mm DTT - 10 mm ATP - 52 ng/μl prepared pet vector - T4 DNA ligase, diluted (with ligase dilution buffer) Weiss units/μl - Prepared target gene insert (0.2 pmol) - Nuclease-free water - Ligase recognition sequences for Not I and Sal I restriction enzymes. Thus, DNA inserts may be cleaved from the vector using EcoR I or either of the rare cutting enzymes, Not I or Sal I. Adapter sequence: 5'-pGTCGACGCGGCCGCG CAGCTGCGCCGGCGCTTAA-OH-5' Storage Buffer 10 mm Tris-HCl (ph 7.5) 100 mm NaCl 1 mm EDTA Quality Control Assays: This product has passed a self-ligation quality control assay. Doc. Rev.:

20 Procedures: One consistently successful protocol for ligation is presented here. 1. For a standard reaction using DNA fragments with 2 4 base sticky ends, use ng ( pmol) of pet vector with 0.2 pmol insert (50 ng of a 500 bp fragment) in a volume of 02 μl. Assemble the following components in a 0.5 ml tube (these components are available separately in the DNA Ligation Kit, Cat. No ) or use the Clonables 0X Ligation Premix (Cat. No ). Add the ligase last. 0 μl 02X Ligase Buffer (022 mm Tris-HCl ph 7.6, 100 mm MgCl2 0 μl 022 mm DTT 0 μl 02 mm ATP 0 μl 52 ng/μl prepared pet vector 0 μl T4 DNA ligase, diluted (with ligase dilution buffer) Weiss units/μl x μl Prepared target gene insert (2.0 pmol) y μl Nuclease-free water to volume 02 μl Total volume 2. Add the ligase last, and gently mix by stirring with a pipet tip. Incubate at 16 C for 2 h to overnight. Also set up a control reaction in which the insert is omitted to check for nonrecombinant background. Note: For blunt ends, use 10X more ligase (i.e., undiluted enzyme), reduce the ATP concentration to 0.1 mm and incubate for 6 16 h at 16 C or 2 h at room temperature. Reagent: Transformation INTO EXPRESSION HOST - competent cell. - SOC Medium. - LB agar plates Procedures: 1. Remove the appropriate number of competent cell tubes from the freezer (include one extra sample for the Test Plasmid positive control, if desired). Immediately place the tubes on ice, so that all but the cap is surrounded by ice. If the standard cells are to be used, place the required number of empty 1.5 ml polypropylene microcentrifuge tubes on ice to prechill. Allow the cells to thaw on ice for ~2 5 min. 2. Visually check the cells to see that they have thawed and gently flick the cells 1 2 times to evenly resuspend the cells. 3. Standard Competent Cells: 20

21 Pipet 02 μl aliquots of cells into the pre-chilled tubes. Singles Competent Cells: Proceed to Step 4 or 5, depending on whether a Test Plasmid sample is included as a positive control. 4. (Optional) to determine transformation efficiency, add 2.0 ng (0 μl) Test Plasmid provided with Competent Cells to one of the tubes containing cells. Stir gently to mix and return the tube to the ice. 5. Add 0 μl of a ligation reaction or purified plasmid DNA directly to the cells. Stir gently to mix and return the tube to the ice, making sure that the tube is surrounded by ice except for the cap. Repeat for additional samples. 6. Incubate the tubes on ice for 5 min. 7. Place the tubes in a 42 C water bath for exactly 30 sec; do not shake. 8. Place the tubes on ice for 2 min. 9. Standard Competent Cells: Add 82 μl of room temperature SOC Medium to each tube. Keep the tubes on ice until all have received SOC. Singles Competent Cells Add 052 μl of room temperature SOC Medium to each tube. Keep the tubes on ice until all have received SOC. 10. Selection for transformation is accomplished by plating on medium containing antibiotic for the plasmid encoded drug resistance. Additional host-specific antibiotics may also be appropriate to ensure maintenance of the host feature(s). When using NovaBlue: if selecting for β-lactamase (carbr/ampr), no outgrowth (shaking incubation) step is required, although slightly higher cloning efficiencies may be obtained with min outgrowth. Plate 5 52 μl cells directly on selective media. If selecting for kanamycin resistance, shake at 37 C (250 rpm) for 30 min prior to plating. When using strains other than NovaBlue: shake at 37 C (250 rpm) for 60 min prior to plating. Prepare LB agar plates with appropriate antibiotic ahead of time Notes: The outgrowth incubation is conveniently performed in a shaking incubator using a test tube rack anchored to the shaking platform. Place each transformation tube in an empty 13 mm x 100 mm glass test tube in the rack. The snap-caps on the transformation tubes prevent them from falling to the bottom of the test tubes, and all transformation tubes remain vertical. During the outgrowth (or earlier if omitting outgrowth), place the plates at 37 C. If the plates contain a lot of moisture, place them cover-side up and open the cover ~1/3 of the way to allow the plates to dry for min. If the plates do not need drying, keep them closed and place them cover-side down in the 37 C incubator for ~20 min prior to plating. 11. Spread 5 52 μl of each transformation on LB agar plates containing the appropriate antibiotic for the plasmid and host strain. When plating less than 05 μl, first pipet a pool of SOC onto the plate and then pipet the cells into the SOC. Please see the part 4.6 for additional details on plating technique. 21

22 Important: The appropriate amount of transformation mixture to plate varies with the efficiency of both the ligation and the competent cells. As little as 0 μl will yield several hundred transformants under highly efficient conditions (e.g., with NovaBlue cells giving > cfu/μg). For recombinants in NovaBlue, expect transformants/μg plasmid, depending on the particular insert and the ligation efficiency. When using the Test Plasmid, plate no more than 5 μl (e.g., 5 μl of NovaBlue cells at efficiency) or 02 μl (e.g., 02 μl of cells at efficiency) of the final transformation mix in a pool of SOC on an LB agar plate containing 52 μg/ml carbenicillin or ampicillin (because the Test Plasmid carries the ampr gene). 12. Let the plates sit on the bench for several min to allow excess liquid to be absorbed, and then invert and incubate overnight at 37 C. 3.3 Extraction and solubilization of inclusion bodies Reagent: - 10mM tris HCl, ph8-10 mm tris HCl, ph7-8m Urea, 100 mm Tris HCl, ph8-8m urea 20 mm Tris,PH 8 - LYSOSYM (100 µg /ml) - Phenyl methyl sulfonyl fluorid (50µg/ml) - DNase (20µg/ml) - RNase (20µg/ml) - 1mM EDTA mm NaCl Purification of recombinant proteins: 1- The cell were harvested by centrifugation at an OD650 OF The cell pellets were resuspended in 10 mm Tris HCl.pH 8 (20 ml) resuspension, containing lysozyme (100 µg / ml), phenyl methyl sulfonyl fluoride (50 µg / ml), DNase(20 µg/ml),rnase (20 µg / ml),and 1mM EDTA and incubated at 00 c for 02 min. 3- The cells were lysed by sonication 13 and then centrifuged (10000xg).for 20 min. 4- The pellet containing recombinant protein was washed with 10 mm Tris HCl, ph 8(20 ml). 13 Sonication is the act of applying sound (usually ultrasound) energy to agitate particles in a sample. In biological applications, sonication may be sufficient to disrupt or deactivate a biological material. This process is called sonoporation. Sonication is also use to fragment molecules of DNA. This is an alternative to the freeze-pump-thaw(by Schlenk flask) and sparging methods. It is especially useful when it is not possible to stir the sample, as with NMR tubes 22

23 5- It is then dissolved in 100 mm Tris HCl, ph8/8 M urea (10 ml), and centrifuged at 4 C for 1 hour.(150000xg) 6- The recombinant protein purified on Q Sepharose fast flow 14x. Using a HiTrap 1-ml column with a syringe. (A) Prepare buffers and sample. Remove the column s top cap and snap off the end. Wash and equilibrate. (B) Load the sample and begin collecting fractions. (C) Wash, elute, and continue collecting fractions fractions, the purified HLA heavy chains contained stored at-20 C for use in the ELISA experiment 3.4 ELISA assay of peptide-mhc complex formation: Material: - Pan specific mouse anti HLA class I antibody,w6/32 - Polyclonal rabbit antihuman β0m-hrp - dextran polymer conjugated with goat antirabbit IgG and HRP - 96 well Maxisorp ELISA Plates - Pluronic lutrol F-68 - Peptide - 100mM carbonate buffer (PH 9.6) - 10 w/v skimmed milk powder in PBS (SMP-PBS) Tween-20 in PBS mm Tris-maleat buffer( PH 6.6) Day 1-96 well Maxisorp ELISA Plates were coated and let overnight at 4 C with W6/ The ion exchange chromatography Q sepharose FF was purchased from GE Healthcare. see for more details 15 Took from GE Healthcare. 23

24 Using 52 μl/well at 5 μl/ml, in 022 mm carbonate buffer, PH 9.6. Day 2 - Add 302 μl/ well 02 w/v skimmed milk powder in PBS ( SMP-PBS) to block the residual bind. - Wash twice with 622 μl/ well of 2.25 Tween-20 in PBS at room temperature using an automated plate washer to remove unbound W6/32 and blocking reagent - On ice, purified recombinant HLA molecule in 8M Urea and 20mM tris PH8 were diluted 100-fold into a 2.3 mm tris maleat buffer, PH 6.6, contaning human β0m, peptide and lutrol F- 68 at the concentrations indicated. 3nM MHC-I HC is optimal 1g/L lutrol F nM β0m is optimal 02,222nM petid (optimal 0nM to 0μM) - To allow complex formation the reaction mixtures were incubated at 18 C for 48h. Day 3 Incubation Day 4 - Just prior to the ELISA analysis, the reaction volume was diluted 10 times into 2 SMP/PBS at 4 C μl/well with PBS/2.25% Tween 02 were transferred in triplicate to a W6/30 coated plate. - The plate was incubated for 2h at 4 C. - Washed 6 times with 622 μl/well with PBS/2.25% Tween 02 at room temperature. - detect the binding complex,the plate was incubated for 1h at 4 c, with 50 µl/ well of a polyclonal rabbit antihuman β0m-hrp diluted 1:2500 into 2% SMP-PBS and the washed 6*600 µl/well with PBS 0.05% tween 20 at room temperature - To enhance the detection, the plate was subsequently incubated for 30 min at room temperature with a dextran polymer conjugated with goat antirabbit IgG and HRP diluted in 1:15 in 2% SMP-PBS containing 1% normal mouse serum. - Washed 6*622 μl/well with PBS/2.25% Tween 02 at room temperature. - ELISA was developed with 3,3 5,5 -tetramethylbenzidine hydrogenperoxide for 30 min at room temperature. 24

25 - Colorimetric reaction was read at 450nm using a Victor Multilabel ELISA counter 25

26 3.5 More detials: I- The peqgold total RNA Kit INTRODUCTION The peqgold Total RNA Kit provides a rapid and easy method for the isolation of up to 100 μg of total RNA from eukaryotic cells and tissues. This kit allows processing of a single or multiple samples in less than 30 min. Normally, up to 1 x 107 cells or 40 mg tissue can be used in a single experiment. There is no need for phenol/chloroform extractions and time-consuming steps such as CsCl gradient ultracentrifugation or precipitation with isopropanol. While this kit may be used for isolation of RNA from whole blood, we recommend to use the peqgold Blood RNA Kit (product # ) as it is specifically designed for effective hemolysis and hemoglobin removal and gives therefore higher RNA yields from blood. RNA purified using the peqgold Total RNA Kit is ready for applications such as RT-PCR, Northern blotting, poly(a)+-rna (mrna) purification, nuclease protection assays and in vitro translation. peqgold Total RNA Kits are available with Safety-Line (Order No xx) or Classic Line (Order No xx) columns. Safety-Line columns can be closed tightly by lids to avoid cross-contamination more effectively. Classic-Line colums do not have lids for a more comfortable handling. PRINCIPLE The peqgold Total RNA Kit uses the reversible binding properties of the PerfectBind RNA Column, a new silica-based material. This is combined with the speed of minicolumn spin technology. A specifically formulated high salt buffer system allows more than 022 μg of RNA molecules greater than 200 bases to bind to the matrix. Cells or tissues are first lysed under denaturing conditions that practically inactivate RNases. Samples are then applied to the PerfectBind RNA Columns to which total RNA binds, while cellular debris and other contaminants are effectively washed out. High quality RNA is finally eluted in RNase-free sterile water. KIT COMPONENTS peqgold Total RNA Kit Order No. Safety-Line Order No. Classic-Line Components PerfectBind RNA Columns DNA Removing Columns 2.0 ml Collection Tubes RNA Lysis Buffer T RNA Wash Buffer I RNA Wash Buffer II (conc.) 26

27 RNase-free Water Instruction manual STORAGE AND STABILITY The peqgold Total RNA Kit components should be stored at room temperature. If stored under these conditions, all components are stable for at least 12 months from the date of purchase. During shipment crystals may form in the RNA Lysis Buffer T. Warm up to 37 C to dissolve. BEFORE STARTING Briefly examine this booklet and become familiar with each step. Prepare all components and have the necessary materials ready before starting.! Whenever working with RNA, always wear one-way gloves to minimize RNase contamination. Use only fresh RNase-free disposable plastic pipette tips when using the supplied reagents.! Work carefully but as quickly as possible during the procedure.! Under cool ambient conditions, crystals may form in RNA Lysis Buffer T. This is normal and the bottle should be warmed (37 C) to dissolve the salt before use.! RNA Wash Buffer II is concentrated and has to be diluted with absolute ethanol as follows: Add 8 ml 100% EtOH to 2 ml Wash Buffer II Add 80 ml 100% EtOH to 20 ml Wash Buffer II Add 3 x 80 ml 100% EtOH to 3 x 20 ml Wash Buffer II.! Store diluted RNA Wash Buffer II at room temperature.! All steps must be carried out at room temperature (22 25 C). PEQGOLD TOTAL RNA ISOLATION PROTOCOL Eucaryotic cells and tissue Materials to be supplied by the user:! 100 % Ethanol! 70 % Ethanol in sterile RNase-free dh2o! Sterile RNase-free pipet tips and centrifuge tubes 1. Homogenization and lysis a. Tissue Excise tissue (~ 40 mg, 3 mm3) and promptly freeze in a small volume of liquid nitrogen. Grind tissue with a ceramic mortar and pestle under approximately 10 ml of liquid nitrogen. Wear gloves and take great care when working with liquid nitrogen. Transfer the suspension into a pre-cooled 15 ml polypropylene tube. If the tube is not precooled (in liquid nitrogen), the suspension will boil vigorously possibly causing loss of tissue. When the liquid nitrogen has completely evaporated, add 422 μl RNA Lysis Buffer T. Transfer the lysate directly into a DNA Removing Column placed in a 2.0 ml Collection Tube. Centrifuge at x g for 1 min at room temperature. Transfer the flow-through lysate into a new 1.5 ml tube. 27

28 For RNase rich tissues or more than 40 mg tissue, use 600 μl of RNA Lysis Buffer T. However, do not use more than 50 mg tissue. For homogenization, you may also use glass-, teflon- or electric homogenisators. b. Monolayer Cells For tissue culture cells grown in monolayer (adherent fibroblasts, endothelial cells etc.), lyse the cells directly in the culture vessel as follows. Aspirate culture medium completely and add RNA Lysis Buffer T directly to the cells. Use 822 μl for T35 flasks or 10 cm dishes, and 422 μl for smaller vessels. Pipet buffer over entire surface of vessel to ensure complete lysis. Transfer the lysate directly into a DNA Removing Column placed in a 2.0 ml Collection Tube. Centrifuge at x g for 1 min at room temperature. Transfer the flow-through lysate into a new 1.5 ml tube. This method is preferable to trypsinization followed by washing because it minimizes RNA degradation by nuclease contamination. c. Suspension culture For cells grown in suspension cultures, pellet cells at rpm (400 x g) for 5 min. Pour off supernatant and add 422 μl RNA Lysis Buffer T per 0 x 027 cells. Transfer the lysate directly into a DNA Removing Column placed in 2.0 ml Collection Tube. Centrifuge at x g for 1 min at room temperature. Transfer the flow-through lysate into a new 1.5 ml tube. 2. Load and Bind Add an equal volume (422 μl, 622 μl or 822 μl) 72 % Ethanol to the lysate and mix thoroughly by vortexing. Place a PerfectBind RNA Column in a new 2.0 ml Collection Tube (supplied) and add the lysate directly to the membrane. Centrifuge the PerfectBind RNA Column / Collection Tube assembly at x g for 1 min. Discard the flowthrough liquid and Collection Tube. A precipitate may form on addition of 70 % ethanol. Vortex and add the entire mixture to the column. The maximum capacity of the PerfectBind RNA Column is 750 μl, larger volumes can be loaded successively. However, the total binding capacity of a PerfectBind RNA Column is approx. 100 μg RNA. 3. Wash I Place the PerfectBind RNA Column in a fresh 2.0 ml Collection Tube, add 522 μl RNA Wash Buffer I to the PerfectBind RNA Column and centrifuge for 15 sec at x g. (supplied). Discard the flow-throw liquid and reuse the collection tube in the next step. 4. DNase Digestion (optional) Since PerfectBind RNA Column technology actually removes most of DNA without a DNase treatment, it is not necessary to do DNase digestion for most downstream applications. However, certain sensitive RNA applications might require further DNA removal. Following steps provide on-membrane DNase I digestion (Order No ). a. For each PerfectBind RNA Column, prepare this DNase I digestion reaction mix: DNase I Digestion Buffer 73.5 μl, RNase-free DNase I (02 Kunitz units/μl) 0.5 μl, Total volume 75.2 μl. Note: 28

29 1. DNase I is very sensitive to physical denaturation, so do not vortex this DNase I mixture! Mix gently by inverting the tube. Prepare fresh DNase I digestion mixture directly before RNA isolation. 2. DNase I digestion buffer is supplied with RNase-free DNase set. Standard DNase buffers are not compatible with on-membrane DNase digestion! b. Pipet 75 μl of the DNase I digestion reaction mix directly onto the surface of PerfectBind RNA resin in each column. Make sure to pipet the DNase I digestion mixture directly onto the membrane. DNase I digestion will not be complete if some of the mix stick to the wall or the O-ring of the PerfectBind RNA Column. c. Incubate at room temperature (25 30 C) for 15 minutes. d. Place the PerfectBind RNA Column into a 0.2 ml Collection Tube and add 422 μl RNA Wash Buffer I. Incubate the PerfectBind RNA Column at benchtop for 5 minutes. Centrifuge at x g for 5 minutes and discard flow-through. Re-use collection tube in the next step. Continue with step Wash II Add 622 μl completed RNA Wash Buffer II to the PerfectBind RNA Column and centrifuge for 15 sec at x g. Discard the flow-through liquid. Repeat this wash step and discard the flow-through liquid. 6. Dry (Important! Do not skip this step!) Place the PerfectBind RNA Column containing your RNA in the collection tube used in step 5 and centrifuge for 1 min at x g to completely dry the column matrix. This step is essential to remove ethanol from the column. 7. Elution Place the PerfectBind RNA Column (step 6) into a fresh 1.5 ml microcentrifuge tube. Add μl (depending on the desired final concentration of RNA) sterile RNAse-free dh2o directly to the binding matrix in the PerfectBind RNA Column and centrifuge for 1min at x g to elute RNA. A second elution may be necessary if the expected yield of RNA is > 50 μg. Alternatively, RNA may be eluted with a higher volume of water. While an additional elution increase total RNA yield, the concentration will be lowered since more than 80 % of RNA is recovered with the first elution. Preheating RNase-free dh2o to 70 C before adding to the PerfectBind RNA Column and incubating the PerfectBind RNA Column for 5 min at room temperature before centrifugation may increase yield. DNA CONTAMINATION No RNA extraction procedure can completely remove genomic DNA. For sensitive work (such as RT-PCR or differential display) we suggest that you treat the eluted RNA with RNase-free DNase. On-membrane DNase I Digestion is a simple and fast method and can be integrated into the standard protocol between the washing steps (see page 14/15). Also for RT-PCR, use intron-spanning primers that allow easy identification of DNAcontamination. 29

30 A PCR reaction, which uses the RNA as template, will also allow the detection of DNA contamination. QUANTITATION AND STORAGE OF RNA Determine the absorption of an appropriate dilution (10- to 50-fold) of the sample at 260 nm and 280 nm. RNase-free water is slightly acidic and can dramatically lower absorption values. We suggest that you dilute the sample in a buffered solution (TE) for spectrophotometric analysis. One A260-unit is about 42 μg RNA/ml. The RNA concentration is calculated as follows: RNA conc. (μg /ml) = Absorption Dilution Factor The ratio of A260/280 is an indication of nucleic acid purity. A value higher than 1.8 indicates > 90 % nucleic acid. Store RNA samples at 70 oc in sterile RNase-free dh2o. Under such conditions RNA prepared with the peqgold system is stable for at least one year. RNA QUALITY It is highly recommended to determine the RNA quality prior to further applications. Denaturing agarose gel electrophoresis and ethidium bromide staining can best assess the quality of RNA. Two sharp bands should appear on the gel. These represent the 28S and 18S ribosomal RNA bands. If these bands smear towards lower molecular weight RNA, then the RNA has undergone major degradation during preparation, handling or storage. Although RNA molecules less than 200 bases in length do not efficiently bind to the PerfectBind RNA Column, a third RNA band, the trna band, may be visible when a large number of cells are used. 30

31 B- Part II The protocol that will be practiced in MEGBI lab contains many modifications for the protocol of part I to fit with the materials and the devices presented in the lab. 3.6 Amplification of the HLA-A*0201 and β-2m gene from PBMC Reagent: peqgold kit. Forward primer for HLA-A*0201: 5 -CCCTGACCCAGACCTGGGCGG-3 Reverse primer for HLA-A*0201: 3 -AGGGTCGGGTGGTAGGGGTAG-5 Forward primer 3 for HLA-A*0201: 5 -GGCTCCCACTCCATGAGGTAT-3 Reverse primer 4 for HLA-A*0201: 3 -GGGAGTGGGACTCTACCCTCG -5. Forward primer for β0-m: 5 -GCATCCAGCGTACTCCAAAGA-3 Reverse primer for β0-m: 3 -ATTCACCCTAGCTCTGTACCG-5 dntp Mix Sterile, distilled water M DTT (200 units) of M-MLV (RT) RNase A 10XPCR Buffer [200mM Tris-HCl (PH 8.4), 500 mm KCl] 50 mm MgCl2 10 mm dntp Mix Amplification primer 1(10µM) Amplification primer 2 (10µM) Taq DNA/polymerase Protocol: 1. Isolate 16 the total RNA that was extracted from the peripheric blood monocytes (PBMC) from 10 ml human_s anticoagulative venous blood was dissolved in 50µL ddh2o. RESULT: the total RNA only 16 The procedures of this step as follows in peqgold kit and for more details see the last page. 31

32 2. Quantify by ultraviolet or spectrophotometer. Goal: for incertitude that find RNA if the number < Amplify the extracellular fragment of HLA-A*0201 (including the fragment of transmembrane) using total RNA as a template with the forward primer: 5 - CCCTGACCCAGACCTGGGCGG-3 and the reverse primer 3 - AGGGTCGGGTGGTAGGGGTAG-5.by PCR as follows: a. Add the following components to a nuclease-free microcentrifuge tube: - Take 0.6µl of the Primer. - Take 5µl of the total RNA. - 1µl (10mM) dntp Mix (10mM each datp,dgtp,dctp and dttp at neutral PH). - Add sterile, distilled water to 12 µl. b. Heat mixture to 65 o C for 5 min and quick chill on ice. C. collect the content of the tube by brief centrifugation (e.g. 20s, rpm) And add: - 4µl 5X First-Strand Buffer 17-2µl 0.1 M DTT c. Mix contents of the tube gently and incubate at 37 o C for 2 min 18 d. Add 1µl (200 units) of M-MLV (RT), and mix by pipetting gently up and down. e. Incubate 50 min at 37 o C. f. Inactivate the reaction by heating at 70 o C for 15 min. g. Incubate 40s at 90 o C (with PCR machine) 17 DTT, Molecular Grade, is an antioxidant used to stabilize enzymes and other proteins containing sulfhydryl groups. The liquid form of the product is a 100mM solution of DTT in water(promega) 18 take the tube of M-MLV out and put in ice until thawed 32

33 h. Immediately put the tube into ice for 1 min i. Add 3µl RNase A j. Incubate 20 min at 37 o C (with PCR machine) Now we have cdna. 4-Add the above to a PCR reaction tube for a final reaction volume of 50 µl: 2.5µl 10 Reaction buffer S 2.5µl 10 Reaction buffer Y 10µl enhancer solution 1µl 40 mm dntp Mix 1µl amplification primer 1(10µM) 1µl amplification primer 2 (10µM) 0.5 µl Taq DNA/polymerase (5U/µl) 2µl cdna (from first-strand reaction) Autoclaved, distilled water to 50 µl Mix gently and layer 1-2 drops (50µl) of silicone oil over the reaction (Note: the addition of silicone oil is unnecessary in thermal cyclers equipped with a heated lid.) Heat reaction to 94 o C for 2 min to denature. PCR program: 35 cycles: 10s 95 o C 10s 60 o C 30-60s 72 o C Take 5µl from the result and put in the second round PCR and repeat the same step. (Note: we use inner primers 3 and 4 in the second round of PCR) 5-Put the PCR product on the gel agarose. 33

34 RESULT: Amplification the DNA segment of HLA-A*0201 only. Analogously, we constructed β-2m expression vector; however, there were some differences between them. The fragment encoding β -2m was amplified using total RNA as a template with the forward primer 5 -GCATCCAGCGTACTCCAAAGA-3 and the reverse primer 3 - ATTCACCCTAGCTCTGTACCG-5. The PCR protocol was the same as that used in amplification of HLA-A*0201. RESULT: Amplification the DNA segment of β2m only. Purify from an agarose gel by QIA quick gel extraction kit 19xi. The DNA sequences were verified by sequencing. 3.7 Prepare the insert DNA (HLA-A*0201 and β2m) with the adapter Suggested Conditions for Adapter Addition to DNA 20. Reagent: 5X adapter buffer EcoR I(Not I) adapter (1 mg/ml) 0.1 M DTT T4 DNA ligase (1 U/μl) T4 polynucleotide kinase. 1. Add the following reagents to a microcentrifuge tube on ice. Up to 5 μg blunt-ended DNA 10 μl 5X adapter buffer (330 mm Tris-HCl (ph 7.6), 50 mm MgCl2, 5 mm ATP) 10 μl EcoR I(Not I) adapter (1 mg/ml) See MEGBI training courses book part I page This step was extracted from invitrogen life technologies. 21 EcoR I(Not I) adapter may be used to add EcoR I cohesive ends directly to any blunt-ended DNA fragment. The adapter is formed by annealing two oligonucleotides together forming a phosphorylated, blunt end and a nonphosphorylated EcoR I half-site. Ligation to blunt ended DNA results in EcoR I half-sites on both ends of the DNA. The adapted DNA may be ligated into any EcoR I containing vector. The adapter also contains the recognition sequences for Not I and Sal I restriction enzymes. Thus, DNA inserts may be cleaved from the vector using EcoR I or either of the rare cutting enzymes, Not I or Sal I. Adapter sequence: 5'-pGTCGACGCGGCCGCG CAGCTGCGCCGGCGCTTAA-OH-5' Storage Buffer 34

35 7 μl 0.1 M DTT distilled water sufficient to bring the volume to 45 μl. 2. Then add 5 μl of T4 DNA ligase (1 U/μl) and mix gently. 3. Incubate the reaction for a minimum of 16 h at 16 C. 4. Heat the reaction at 70 C for 10 min to inactivate the ligase. 5. Place the reaction on ice. The adapted DNA, after adapter removal (step 9) may be ligated directly to any phosphorylated, EcoR I-digested vector. However, if a dephosphorylated EcoR I-digested vector is used, the adapted cdna must be phosphorylated first before adapter removal (step 6). 6. Add 3 μl (30 units) of T4 polynucleotide kinase 22 to the reaction from step Mix gently, and incubate the reaction for 30 min at 37 C. 8. Heat the reaction at 70 C for 10 min to inactivate the kinase and place the reaction on ice. 9. Remove the excess EcoRI (NotI) adapters by gel electrophoresis and then ligate into the appropriate vector. Prepare the T7 RNA polymerase with the adapter The protocol was the same as that used in preparation of insert DNA with the adapter. 3.8 Expression of HLA-A*0201 and β-2m Vector preparation: To digest and gel-purify the vector: Reagent: pet vector 10X restriction enzyme buffer EcoR I restriction enzyme 10 mm Tris-HCl (ph 7.5) 100 mm NaCl 1 mm EDTA Quality Control Assays: This product has passed a self-ligation quality control assay. Doc. Rev.: T4 Polynucleotide Kinase (T4 PNK) catalyzes the transfer of the γ-phosphate from ATP to the 5 -terminus of polynucleotides or to mononucleotides bearing a 3 -phosphate group (1). T4 PNK is widely used to end-label short oligonucleotide probes (2), DNA (3) and RNA (4) molecules (Promega) 35

36 1. Assemble the following components in a microcentrifuge tube: 3 μg pet vector 3 μl 02X restriction enzyme buffer U EcoR I restriction enzyme (assuming compatible buffer; the total volume of enzyme added should not exceed 10% of the reaction volume to avoid high glycerol concentrations) x μl Nuclease-free water brought to volume 32 μl Total volume 2. Incubate at the appropriate temperature (usually 37 C) for 2 4 h. 3. Run a 3 μl sample on an agarose gel to check the extent of digestion. 4. When digestion is complete, add calf intestinal alkaline phosphatase (Calbiochem Cat. No ) directly to the remainder of the digestion. The enzyme functions in most restriction buffers under the conditions described here. It is important to use the correct amount of enzyme; too much can cause unwanted deletions and can be difficult to remove for future steps. Three μg of a typical pet vector (5 kbp) corresponds to about 2 pmol DNA ends when linearized, or about 4 pmol ends if two enzymes were used for digestion. We recommend using 0.05 units of alkaline phosphatase per pmol ends. Dilute the enzyme in water or 50 mm Tris- HCl, ph 9.0 just before use. 5. Incubate at 37 C for 30 min. 6. Add gel sample buffer to the reaction and load the entire sample into a large well ( cm wide) on a 0% agarose gel containing 2.5 μg/ml ethidium bromide. Run the gel far enough to separate the linear plasmid from nicked and supercoiled species. It is useful to run uncut vector DNA in an adjacent lane to help distinguish undigested from linearized plasmid DNA. 7. Visualize the DNA band with a long wave UV light source and cut the band from the gel using a clean razor blade. Avoid over exposure to the light source, which can cause nicks and double strand breaks in the DNA. 8. Recover the DNA from the gel slice by purification with QIA quick gel extraction kit 23xii Resuspend the final product in a total volume of 32 μl (usually about 52 ng/μl DNA). Assume recoveries in the range of 50% for the ligation step. 9. Store the treated vector at 20 C until use. 23 Refer to MEGBI training courses book part I page

37 3.8.2 Ligation N.B: this step should be done in 3 eppendorfs tubes for HLA-A*2020, β0-m and T7 RNA polymerase. Reagent: 10X Ligase Buffer (200 mm Tris-HCl ph 7.6, 100 mm MgCl2 100 mm DTT 10 mm ATP, (or 2 rapid ligation buffer contains ATP) 52 ng/μl prepared pet vector T4 DNA ligase, diluted (with ligase dilution buffer) Weiss units/μl Prepared target gene insert (0.2 pmol) Nuclease-free water Procedures: 1. For a standard reaction using DNA fragments with 2 4 base sticky ends, use ng ( pmol) of pet vector with 0.2 pmol insert (50 ng of a 500 bp fragment) in a volume of 02 μl. Assemble the following components in a 1.5 ml tube: 0 μl 02X Ligase Buffer (022 mm Tris-HCl ph 7.6, 100 mm MgCl2 0 μl 022 mm DTT 0 μl 02 mm ATP 0 μl 52 ng/μl prepared pet vector 0 μl T4 DNA ligase, diluted (with ligase dilution buffer) Weiss units/μl x μl Prepared target gene insert (0.2 pmol) y μl Nuclease-free water to volume 02 μl Total volume 2. Gently mix by stirring with a pipet tip. Incubate at 16 C for 2 h to overnight. Also set up a control reaction in which the insert is omitted to check for nonrecombinant background Transformation Preparation of E.coli competent cells 24. Reagent: Competent cells JM Refer to MEGBI training courses book part I page

38 LB Medium. LB agar plates. Procedures: 1. Remove the appropriate number of competent cell tubes from the freezer (include one extra sample for the Test Plasmid positive control, if desired). Immediately place the tubes on ice, so that all but the cap is surrounded by ice. If the standard cells are to be used, place the required number of empty 1.5 ml polypropylene microcentrifuge tubes on ice to prechill. Allow the cells to thaw on ice for ~2 5 min. 2. Visually check the cells to see that they have thawed and gently flick the cells 1 2 times to evenly resuspend the cells. 3. Standard Competent Cells: Pipet 02 μl aliquots of cells into the pre-chilled tubes. Singles Competent Cells: Proceed to Step 4 or 5, depending on whether a Test Plasmid sample is included as a positive control. 4. (Optional) to determine transformation efficiency, add 2.0 ng (0 μl) Test Plasmid provided with Competent Cells to one of the tubes containing cells. Stir gently to mix and return the tube to the ice. 5. Add 0 μl of each ligation reaction (ligation of HLA-A*0201 and ligation of T7 RNA polymerase) or purified plasmid DNA directly to the cells. Stir gently to mix and return the tube to the ice, making sure that the tube is surrounded by ice except for the cap. Repeat for additional samples. 6. Incubate the tubes on ice for 5 min. 7. Place the tubes in a 42 C water bath for exactly 30 sec; do not shake. 8. Place the tubes on ice for 2 min. 9. Standard Competent Cells: Add 82 μl of room temperature LB medium to each tube. Keep the tubes on ice until all have received LB. Singles Competent Cells Add 052 μl of room temperature LB Medium to each tube. Keep the tubes on ice until all have received LB. 10. Selection for transformation is accomplished by plating on medium containing antibiotic (ampicillin) for the plasmid encoded drug resistance. 38

39 When using strains other than NovaBlue: shake at 37 C (250 rpm) for 60 min prior to plating. Prepare LB agar plates with appropriate antibiotic ahead of time Notes: The outgrowth incubation is conveniently performed in a shaking incubator using a test tube rack anchored to the shaking platform. Place each transformation tube in an empty 13 mm x 100 mm glass test tube in the rack. The snap-caps on the transformation tubes prevent them from falling to the bottom of the test tubes, and all transformation tubes remain vertical. During the outgrowth (or earlier if omitting outgrowth), place the plates at 37 C. If the plates contain a lot of moisture, place them cover-side up and open the cover ~1/3 of the way to allow the plates to dry for min. If the plates do not need drying, keep them closed and place them cover-side down in the 37 C incubator for ~20 min prior to plating. 11. Spread 5 52 μl of each transformation on LB agar plates containing the appropriate antibiotic for the plasmid and host strain. Please see the last page for additional details on plating technique. Important: The appropriate amount of transformation mixture to plate varies with the efficiency of both the ligation and the competent cells. As little as 0 μl will yield several hundred transformants under highly efficient conditions (e.g., with NovaBlue cells giving > cfu/μg). When using the Test Plasmid, plate no more than 5 μl (e.g., 5 μl of NovaBlue cells at efficiency) or 02 μl (e.g., 02 μl of cells at efficiency) of the final transformation mix in a pool of LB on an LB agar plate containing 52 μg/ml carbenicillin or ampicillin (because the Test Plasmid carries the ampr gene). 12. Let the plates sit on the bench for several min to allow excess liquid to be absorbed, and then invert and incubate overnight at 37 C. 3.9 Extraction and solubilization of inclusion bodies Materiel: 10mM tris HCl, ph8 10 mm tris HCl, ph7 8M Urea, 100 mm Tris HCl, ph8 8M urea 20 mm Tris, PH 8 LYSOSYM (100 µg /ml) Phenyl methyl sulfonyl fluoride (50µg/ml) DNase (20µg/ml) 39

40 RNase (20µg/ml) 1mM EDTA mm NaCl Purification of recombinant proteins: Incubation of some colonies overnight with lb medium with ampicillin. Centrifugation at 4000 rpm for 5min. The cell pellets were resuspended in 10 mm Tris HCl.pH 8 (250µl) resuspension, containing lysozyme (100 µg / ml), phenyl methyl sulfonyl fluoride (50 µg / ml), DNase(20 µg/ml),rnase (02 μg / ml),and 0mM EDTA and incubated at 00 c for 02 min. The cells were lysed by heat shock for 40s in boiling water and then centrifuged (10000xg) for 20 min. The pellet containing recombinant protein was washed with 10 mm Tris HCl, ph 8(250µl). It is then dissolved in 100 mm Tris HCl, ph8/8 M urea (125µl), and centrifuged at 4 C for 1 hour (150000xg) The recombinant protein purified by an ion exchange chromatography Q Sepharose fast flow 25. Fractions, the purified HLA heavy chains contained stored at-20 C for use in the ELISA experiment 3.10 ELISA assay of peptide-mhc complex formation: Material: - Pan specific mouse anti HLA class I antibody, W6/32 - Polyclonal rabbit antihuman β0m-hrp or post primary block (from Novolink compact polymer Detection kit) - dextran polymer conjugated with goat antirabbit IgG and HRP or Novolink polymer (from Novolink compact polymer Detection kit) - 96 well Maxisorp ELISA Plates - Pluronic lutrol F-68 - Peptide - 100mM carbonate buffer (PH 9.6) 25 The ion exchange chromatography Q sepharose FF was purchased from GE Healthcare. see for more details 40

41 - 10 w/v skimmed milk powder in PBS (SMP-PBS) or Protein block (from Novolink compact polymer Detection kit) Tween-20 in PBS mm Tris-maleat buffer (PH 6.6) Day 1-96 well Maxisorp ELISA Plates were coated and let overnight at 4 C with W6/32. Using 52μl/well at 5μl/ml, in 020 mm carbonate buffer, PH 9.6. Day 2 Add 302 μl/ well 02 w/v protein block to block the residual bind. Wash twice with 622 μl/ well of 2.25 Tween-20 in PBS at room temperature using an automated plate washer to remove unbound W6/32 and blocking reagent On ice, purified recombinant HLA molecule in 8M Urea and 20mM Tris PH8 were diluted 100- fold into a 0.3 mm Tris maleat buffer, PH 6.6, containing human β0m, peptide and lutrol F- 68 at the concentrations indicated: 3nM MHC-I HC is optimal 1g/L lutrol F nM β0m is optimal 10,000nM peptide (optimal 0nM to 0μM) To allow complex formation the reaction mixtures were incubated at 18 C for 48h. Day 3 Incubation Day 4 Just prior to the ELISA analysis, the reaction volume was diluted 10 times into 2 protein block at 4 C. 52 μl/well with PBS/2.25% Tween 02 were transferred in triplicate to a W6/30 coated plate. The plate was incubated for 2h at 4 C. Washed 6 times with 622 μl/well with PBS/2.25% Tween 02 at room temperature. 41

42 detect the binding complex, the plate was incubated for 1h at 4 c, with 50 µl/ well of a post primary bock with 2% protein block and the washed 6*600 µl/well with PBS 0.05% Tween 20 at room temperature To enhance the detection, the plate was subsequently incubated for 30 min at room temperature with. Novolink polymer. Washed 6*622 μl/well with PBS/2.25% Tween 02 at room temperature. ELISA was developed with 3, 3 5, 5 -tetramethylbenzidine hydrogenperoxide 26 for 30 min at room temperature. Count spots by eye 27xiii, or Count spots using a dissecting microscope or automated ELISPOT plate reader. Store plates in the dark prior to reading 28 or wrap in foil and store at -20 C until reading 29xiv ELISA buffers. ELISA Wash Buffer: 1X PBS with 0.05% Tween-20 (0.5 ml Tween-20 in 1 L PBS) 1X PBS: 80.0 g NaCl 11.6 g Na2HPO2 2.0 g KH2PO4 2.0 g KCl Qs with DI H20 up to 10.0 L, ph to For more details: The peqgold viral RNA Kit INTRODUCTION The peqgold viral RNA Kit is designed for isolation of viral RNA from cell free fluids such as plasma, serum, urine and cell culture supernatants. The kit is also suitable for isolation of total RNA from cultured cells, tissues and bacteria. 26 Ready-to-use sensitive substrate for the detection of horseradish peroxidase activity. Absorbs at 450 nm (yellow end-product). Ideal for ELISA and solution assays. 27 peptides@thinkpeptides.com Web:

43 RNA purified using the peqgold viral RNA Kit method is ready for applications such as RT- PCR Protocol: Purification of Viral RNA (Spin Protocol) This protocol is for purification of viral RNA from 140 μl plasma, serum, urine, cell culture. Important points before starting Read Important Notes (pages 30) All centrifugation steps are carried out at room temperature (15 25 C). Things to do before starting Equilibrate samples to room temperature (15 25 C). Equilibrate Buffer AVE to room temperature for elution in step 11. Check that Buffer AW1 and Buffer AW2 have been prepared according to the instructions on page 32. Add carrier RNA reconstituted in Buffer AVE to Buffer AVL Important; if not have QIAamp Viral RNA mini, we can use peqgold Viral RNA Kit protocoles visit procedure 1. Pipet 560 μl of prepared Buffer AVL containing carrier RNA into a 1.5 ml microcentrifuge tube. If the sample volume is larger than 140 μl, increase the amount of Buffer AVL carrier RNA proportionally (e.g., a 280 μl sample will require 1120 μl Buffer AVL carrier RNA) and use a larger tube. Spin Protocol * Fully automatable on the QIAcube. See for protocols. 2. Add 140 μl plasma, serum, urine, cell-culture supernatant, or cell-free body fluid to the Buffer AVL carrier RNA in the microcentrifuge tube. Mix by pulse-vortexing for 15 s. To ensure efficient lysis, it is essential that the sample is mixed thoroughly with Buffer AVL to yield a homogeneous solution. Frozen samples that have only been thawed once can also be used. 3. Incubate at room temperature (15 25 C) for 10 min. Viral particle lysis is complete after lysis for 10 min at room temperature. Longer incubation times have no effect on the yield or quality of the purified RNA. Potentially infectious agents and RNases are inactivated in Buffer AVL. 43

44 4. Briefly centrifuge the tube to remove drops from the inside of the lid. 5. Add 560 μl of ethanol (96 100%) to the sample, and mix by pulse-vortexing for 15 s. After mixing, briefly centrifuge the tube to remove drops from inside the lid. Only ethanol should be used since other alcohols may result in reduced RNA yield and purity. Do not use denatured alcohol, which contains other substances such as methanol or methylethylketone. If the sample volume is greater than 140 μl, increase the amount of ethanol proportionally (e.g., a 280 μl sample will require 1120 μl of ethanol). In order to ensure efficient binding, it is essential that the sample is mixed thoroughly with the ethanol to yield a homogeneous solution. 6. Carefully apply 630 μl of the solution from step 5 to the QIAamp Mini column (in a 2 ml collection tube) without wetting the rim. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini column into a clean 2 ml collection tube, and discard the tube containing the filtrate. Close each spin column in order to avoid cross-contamination during centrifugation. Centrifugation is performed at 6000 x g (8000 rpm) in order to limit microcentrifuge noise. Centrifugation at full speed will not affect the yield or purity of the viral RNA. If the solution has not completely passed through the membrane, centrifuge again at a higher speed until all of the solution has passed through. 7. Carefully open the QIAamp Mini column, and repeat step 6. If the sample volume was greater than 140 μl, repeat this step until all of the lysate has been loaded onto the spin column. 8. Carefully open the QIAamp Mini column, and add 500 μl of Buffer AW1. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini column in a clean 2 ml collection tube (provided), and discard the tube containing the filtrate. It is not necessary to increase the volume of Buffer AW1 even if the original sample volume was larger than 140 μl. Spin Protoco 9. Carefully open the QIAamp Mini column, and add 500 μl of Buffer AW2. Close the cap and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min. Continue directly with step 11, or to eliminate any chance of possible Buffer AW2 carryover, perform step 10, and then continue with step 11. Note: Residual Buffer AW2 in the eluate may cause problems in downstream applications. Some centrifuge rotors may vibrate upon deceleration, resulting in flow-through, containing Buffer AW2, contacting the QIAamp Mini column. 44

45 Removing the QIAamp Mini column and collection tube from the rotor may also cause flowthrough to come into contact with the QIAamp Mini column. In these cases, the optional step 10 should be performed. 10. Recommended: Place the QIAamp Mini column in a new 2 ml collection tube (not provided), and discard the old collection tube with the filtrate. Centrifuge at full speed for 1 min. 11. Place the QIAamp Mini column in a clean 1.5 ml microcentrifuge tube (not provided). Discard the old collection tube containing the filtrate. Carefully open the QIAamp Mini column and add 60 μl of Buffer AVE equilibrated to room temperature. Close the cap, and incubate at room temperature for 1 min. Centrifuge at 6000 x g (8000 rpm) for 1 min. A single elution with 60 μl of Buffer AVE is sufficient to elute at least 90% of the viral RNA from the QIAamp Mini column. Performing a double elution using 2 x 40 μl of Buffer AVE will increase yield by up to 10%. Elution with volumes of less than 30 μl will lead to reduced yields and will not increase the final concentration of RNA in the eluate. Viral RNA is stable for up to one year when stored at 20 C or 70 C. Transformation 30 Initial cloning should be done in a reca cloning strain, such as NovaBlue, or other similar host that lacks the gene for T7 RNA polymerase. This enables high percentage monomer plasmid yields for examination of the construct sequence, as well as separation of cloning from expression. This separation can be valuable in troubleshooting any difficulties that might arise during later procedures. The strains described above for cloning and expression with pet vectors can be prepared for transformation by standard procedures. Expect BL21 (an expression strain) and its derivatives to be transformed at about 1/10 the efficiency of the other strains. For convenience and consistent performance, Novagen offers the relevant host strains as prepared competent cells, ready for high-efficiency transformation. DNA in ligation reactions containing high-quality reagents is suitable for direct addition to Novagen s Competent Cells (no more than 0 μl ligation should be used per 02 μl cells). Inactivation of the ligase is not required prior to transformation. Plasmid DNA isolated using standard preparation procedures is also usually satisfactory; however, for maximum efficiency, 30 Extracted from Novagen 45

46 the sample DNA should be free of phenol, ethanol, salts, protein and detergents, and dissolved in TE buffer (10 mm Tris-HCl ph 8.0, 1 mm EDTA) or in water. Novagen s Competent Cells are provided in 2.0 ml aliquots. The standard transformation reaction requires 02 μl cells, so each tube contains enough cells for 10 transformations. Singles Competent Cells are provided in 52 μl aliquots, which are used as is for single 52 μl transformations. Note that there are a few steps in the protocol that vary for the Singles vs. standard cells. Novagen s NovaBlue and BL00 (DE3) Competent Cells are also offered in a highthroughput 96-well plate format known as HT96 Competent Cells (see Technical Bulletin 313). Handling Tips 1. Upon receipt from Novagen, verify that the competent cells are still frozen and that dry ice is still present in the shipping container. Immediately place the competent cells at 70 C or below. For optimal results, do not allow the cells to thaw at any time prior to use. 2. Handle only the very top of the tube and the tube cap to prevent the cells from warming. Keep the cells on ice whenever possible. 3. To mix cells, flick the tube 1 3 times. NEVER vortex the competent cells. 4. To avoid multiple freeze-thaw cycles of the standard 0.2 ml cells, dispense the cells into aliquots after the initial thaw and store them at 70 C or below (note that Singles Competent Cells are provided as 52 μl aliquots, which are used as is and do not require dispensing. To dispense aliquots of cells from the 0.2 ml stock, remove the stock tube quickly from the ice and flick 1 0 times to mix prior to opening the tube. Remove a 02 μl aliquot from the middle of the cells, and replace the tube immediately on ice. Place the aliquot immediately into the bottom of a pre-chilled 1.5 ml tube, mix by pipetting once up and down, and then immediately close the tube and replace on ice. After all of the aliquots are taken, return any unused tubes to the freezer before proceeding with the transformation. Plating techniques 1. Remove the plates from the incubator. If plating less than 05 μl of the transformation, we recommend plating onto a pool of SOC, which facilitates even colony distribution on the plate surface. Using a sterile pipet tip, place μl of SOC in the center of a plate for a plating cushion. 2. To remove the transformation sample, flick the transformation tube 5 8 times, open the cap and immediately remove the sample volume from the middle of the transformation reaction. 3. Transfer the sample to the plate by dispensing the sample volume into the SOC cushion. 46

47 After the sample is out of the pipet tip, use the same tip to pipet up the sample volume s worth of SOC from the cushion edge and dispense that SOC back into the cushion. (This effectively rinses out your pipet tip.) ColiRollers Plating Beads To use ColiRollers, simply dispense beads per plate. The beads can be dispensed before or after pipetting the transformation mix on the plate. Cover the plate with its lid and move the plate back and forth several times. The rolling action of the beads distributes the cells. Several plates can be stacked up and shaken at one time. After all plates have been spread, discard the ColiRollers by inverting the plate over a collection container. Cover and incubate (step 12 above). ColiRollers Plating Beads are treated glass beads that eliminate the use of the spreader and alcohol flame while evenly and consistently distributing cells without damage. Standard spreader Completely immerse the plating spreader (bent glass rod or equivalent) into ethanol and flame to sterilize. After the flame is extinguished, allow the spreader to cool ~10 sec prior to placing the spreader on the plate. Place the spreader on the LB agar at the outside of the plate (not touching the pool of cells). This further cools the spreader on the LB agar before spreading the cells. Slowly turn the plate while supporting the weight of the spreader. Important: Do not press down on the spreader use just enough pressure to spread the cells. Spread until the sample is evenly distributed on the plate. If the plates are fairly dry, the sample and cushion will quickly absorb into the plate. After the moisture is absorbed, do not continue spreading. If the plates are wet, spread until the sample is evenly distributed. Do not spread until the sample and cushion have absorbed completely into the plate, because overspreading can decrease transformation efficiency. Instead, after spreading briefly, allow the plates to sit upright at room temperature for ~15 min prior to placing them in the 37ºC incubator. This will allow excess moisture to absorb into the plates before the plates are inverted and placed in the incubator. Incubate all plates, cover-side down, in the 37ºC incubator for h. To obtain larger colonies, extend the incubation time slightly (1 2 h), but beware of the potential for development of satellite colonies with extended incubations (usually > 36 h at 37 C; satellites are not commonly observed when using carbenicillin or kanamycin). Once the colonies are at the desired size, the plates can be placed at 4ºC. 47

48 The Host Strains Host Strains 31xv After plasmids are established in a non-expression host, they are most often transformed into a host bearing the T7 RNA polymerase gene ( DE3 lysogen) for expression of target proteins. Figure 1 illustrates in schematic form the host and vector elements available for control of T7 RNA polymerase levels and the subsequent transcription of a target gene in a pet vector. In DE3 lysogens, the T7 RNA polymerase gene is under the control of the lacuv5 promoter. This allows some degree of transcription in the uninduced state and in the absence of further controls is suitable for expression of many genes whose products have innocuous effects on host cell growth. For more stringent control, hosts carrying either plyss or plyse are available. The plys plasmids encode T7 lysozyme, which is a natural inhibitor of T7 RNA polymerase, and thus reduces its ability to transcribe target genes in uninduced cells. plyss hosts produce low amounts of T7 lysozyme, while plyse hosts produce much more enzyme and, therefore, represent the most stringent control available in DE3 lysogens (4). Figure 1. Control elements of the pet system Several different host strains are available as DE3 lysogens. The most widely used host is BL21, which has the advantage of being deficient in both lon (5) and ompt proteases. Novagen has introduced two derivatives of BL21 designed for special purposes. The B834 series is methionine deficient and, therefore, enables high specific activity labeling of target proteins with 35 S- 31 United States & Canada Germany United Kingdom Or your local sales office 48

49 methionine or selenomethionine (6). The BLR strain is a reca- derivative that improves plasmid monomer yields and may help stabilize target plasmids containing repetitive sequences. The AD494 strains are thioredoxin reductase (trxb) mutants that enable disulfide bond formation in the E.coli cytoplasm. This allows for the potential production of properly folded, active proteins (7). Other available strain backgrounds include the K-12 strains HMS174 and NovaBlue, which are reca-, like BLR. These strains may stabilize certain target genes whose products may cause the loss of the DE3 prophage. NovaBlue is potentially useful as a stringent host due to the presence of the high affinity laci q repressor encoded by the F episome. In addition, Novagen offers the DE3 Lysogenization Kit for making new expression hosts with other genetic backgrounds. An alternative for expressing extremely toxic genes or preparing a new DE3 lysogen is to provide T7 RNA polymerase by infection with CE6. Although not as convenient as inducing a DE3 lysogen with IPTG, this strategy may be preferred for certain applications. High Stringency T7lac Promoter In addition to the choice of three basic expression stringencies at the host level, the pet system provides two different stringency options at the level of the T7 promoter itself: the "plain" T7 promoter and the T7lac promoter (8; also shown in Fig. 1). The T7lac promoter contains a 25 bp lac operator sequence immediately downstream from the 17 bp promoter region. Binding of the lac repressor at this site effectively reduces transcription by T7 RNA polymerase, thus providing a second laci-based mechanism (besides the repression at lacuv5) to suppress basal expression in DE3 lysogens. Plasmids with the T7lac promoter also carry their own copy of laci to ensure that enough repressor is made to titrate all available operator sites. In practice, it is usually worthwhile to test several different vector/host combinations to obtain the best possible yield of protein in its desired form (9). Figure 2 illustrates dramatic differences in the expression of two target proteins with various combinations. 49

50 50

51 النتائج / Results 4 مضاعفة جين هال* 2020 ويبتا- / PBMC 4.1 Amplification of the HLA-A*0201 and β-2m gene from 0 آم من المحيط الخارجي لخاليا الدم البيضاء First: we isolated the total RNA form PBMC as follows: We drew 10ml of blood from a person, we putted it in 4ml of EDTA (fig.1) to prevent the agglutination (EDTA is an anticoagulant) and we centrifugated it into a centrifugation at 4000rpm for 8 minutes (fig.2), we obtained the result designed in fig.3 then we aspirated the phase 2 that contains the white blood cells with a micropipette 1000µl, we putted it in a 1.5ml microcentrifuge tube finally we isolated the total RNA by the peqgold viral RNA kit (can be stored the result at 4 C). أوال : قمنا بعزل الرنا الكامل من احمليط اخلارجي خلاليا الدم البيضاء كما يلي: قمنا بسحب 10 مل من دم إنسان ووضعناه يف 4 مل من إيثلني ثنائي األمني رباعي ضبض اخلل )EDTA( )صورة 1( دلنع زبثر الدم )منع التجلط( ومن مث وضعناه يف آلة الطاردة لفصل الدم على سرعة 4000 آر.يب.آم دلدة 8 دقائق )صورة 2( وبعدىا حصلنا على ثالث طبقات من الدم كما يف الصورة 3 وبواسطة ادلاصة األوتوماتيكية اخلاصة ب. 1000µl سحبنا الطبقة الثانية من الدم احملتوية على كريات الدم البيضاء ووضعناىا يف أنبوب 1.5 مل وبدأنا بعملية عزل الرنا الكامل بواسطة رلموعة الرنا الفريوسي بك غولد. )ربفظ النتيجة على 4 مº ( Figure 1: putting the blood in 4ml الاورة 1: وضع 10 مل من الدم في / EDTA 4 مل من مضاد التخثر Figure 2: centrifugation at 4000rpm for 8min / الاورة 2: عملية الفال على 4000 آر. يب.آم لمدة 8 دقائق الاورة / centrifugation Figure 3: Blood components after 3: مكونات الدم بعد الفال. Second: we retrotranscripted the trna to cdna by M-MLV reverse transcriptase as described in part 3.6 ثانيا : قمنا بعملية إعادة نسخ الرنا الناقل إىل الدنا الناسخ بواسطة أنزمي -M MLV reverse transcriptase كما ىو مذكور يف اجلزء 3.6 ادلرحلة 51

52 م step 3. Third: we amplified the cdna by PCR as described in part 3.6 step 4, and we obtained a quantity amplified of HLA- A*2020 and β0-m..3 ثالثا : بعدما حصلنا على الدنا الناسخ قمنا دبضاعفة كميتو بواسطة آلة تفاعل البلمرة ادلتسلسل كما ىو مذكور يف اجلزء 3.6 ادلرحلة 4 وىكذا نكون قد حصلنا علىكمية مضاعفة من ال ىال* 0201 وبيتا 2 -آم. Figure 4: Pipetting of 1µl of M-MLV(RT) / الاورة 4: - Mإمتااص 1 ميكروليتر من MLV Figure 5: PCR machine / آلة تفاعل البلمرة المتسلسل 5: الاورة Fourth: we purified the PCR product on an agarose gel; we prepared the agarose gel as follows: The preparation of an agarose gel: - Put 1g of agarose in 100ml of TAE buffer 1 PH and dissolve it at 90 C. - Let the gel cool down to about 60 C at room temperature. - Put the gel on the gel rack then insert the comb and let it to solidify. رابعا : قمنا بتنقية منتج آلة تفاعل البلمرة ادلتسلسل بواسطة ىالم األغاروز: 1. قمنا بتحضري اذلالم األعاروزيكما يلي: طريقة التحضري: - وضعنا 1 غ من األغاروز يف 100 مل من منظم. 1 درجة TAE احلموضة مث قمنا يتذويبو على 90 - تركناه ليربد إىل 60 مº على درجة حرارة الغرفة. - أضفناه على طبق اذلالم وأضفنا عليو ادلشط مث تركناه حىت جيمد. 52

53 Figure 6: Dissolve the gel at الاورة / agitation 90 C with تذويب الهالم على 90 م مع التحريك. :6 Figure 7: putting of the gel الاورة :7 وضع / rack on the gel الهالم على الطبق Figure 8: injetion of 10µl of sample الصورة :8 وضع / gel into the wells of 10 ميكروليتر من العينة في فتحة الهالم Figure 9: migration of the three bands (2 bands for HLA-A*0201& 1 band for β2-m) الاورة 9: ىجرة البقع الثالثة )بقعتين للهال وبقعة لبيتا 2 -آم( 2. After the solidification of gel, we drew the comb and the border of the gel rack, we added 1liter of TAE buffer to cover all the gel, then we injected 10µl of each sample (we putted in each new eppendorf tube 6 µl of the DNA (HLA- A*2020 or β-2m) + 3µl of glycerol + 3µl of bormophenol blue) into the wells of the gel prepared, we closed the lid of electrophoresis chamber and applied the voltage at 120 volts for 30min, (400mA, 400 Watts). 3. After the migration of bands we turned off the power supply we removed the gel and putted it into a deep vessel we covered it by 250ml of dh2o and we added 30µl of ethidium bromide and late it for 30min. then we washed the gel 3 times from ethidium bromide with dist.water.(250ml in each times). 2. بعد صباد اذلالم جيدا قمنا بسحب ادلشط وأطراف طبق اذلالم وأضفنا 1 لي ت من منظم 1 درجة TAE احلموضة لتغطية اذلالم كامال ووضعنا يف الفتحات الصغرية للهالم 10 ميكرولي ت من كل عينة )ربتوي العينة يفكل أنبوب على 6 ميكرولي ت من الدنا إما اذلال 0201 وإما بيتا 2 -آم + 3 ميكرولي ت من الغليسريول + 3 ميكرولي ت من الربوموفينول األزرق( مث أغلقنا غطاء الرحالن الكهربائي وقمنا بتشغيل التيار الكهربائي على 120 ڤولت دلدة 30 دقيقة. 3. بعد ىجرة بقع الدنا قمنا بإطفاء التيار الكهربائي عن اآللة أخذنا اذلالم ووضعناه يف وعاء عميق أضفنا عليو 250 مل من ادلاء ادلعقم مع 30 ميكرولي ت من بروميد اإلثيديوم وتركناه دلدة 30 دقيقة بعدىا قمنا بغسلو من بروميد اإلثيديوم 3 مرات بواسطة ادلاء ادلعقم )وضعنا يف كل مرة 250 مل من ادلاء ادلعقم(. : be curful with the ethidium bromide!!! Use specific gloves, put the waste liquid of ethidium bromide in a specific place, and do not throw it in the nature. : يجب الحذر في التعامل مع اإلتيديوم!!! إستخدم القفازات الخاصة, ضع الماء المحتوية على بروميد اإلثيديوم في مكان خاص و ال ترميو في الطبيعة. ألنو قد يسبب السرطان ويحدث الطفرات 53

54 Ethidium bromide could cause a cancer and it is mutagen. 4. We putted the gel on UV-machine to see if we have bands or no, if yes, so the steps are correct and we can continue the protocol. Result: AlHamdullillah we observed the 3 bands and we could continue the protocol (fig.12). )التغيير المفاجئ للدنا (. 4. بعد غسلو جيدا من بروميد اإلثيديوم وضعناه على آلة األشعة ما فوق البنفسجية لنرى إذا كان ىناك بقع من الدنا إال إن ىذه ادلرحلة ستبني لنا نتيجة ما مت فعلو حىت اآلن. النتيجة: احلمدهلل سبكنا من رؤية البقع الثالثة اليت مت وضعهم )بقعتني للهال وبقعة لبيتا 2 -آم( وسبكنا من إكمال العمل )الصورة- 12 (. Figure 10:injection of 30µl of ethidium bromde in the gel recovered with 250ml /dh2o. الاورة 10: إضافة 30 ميكروليتر من بروميد اإلثيديوم في الهالم المغطى ب 250 مل من الماء المعقم Figure 11: soaking the gel with /ethidium bromide for 30min الاورة 11: نقع الهالم ببرميد اإلثيديوم لمدة 30 دقيقة Figure 12: observation of 2 bands of HLA-A*0201 at the right an 1 band of β2-m at the left on UVmachine / الاورة 12: مالحظة بقعتي الدنا من الهال 0201 من الجهة اليمنى للهالم وبقعة للبيتا 2 -آم على الجهة اليسرى منو على آلة األشعة ما فوق البنفسجية. 5. We marked off the border of each band on the UV-machine (fig.14), then we putted the gel in the deep vessel to cut the bands and should be remove all the excess of gel (fig.15) then we putted each band in a new eppendorf tube (fig.16), we balanced each tube before and after the addition of band in it for obtain the weight of bands to begin the purification of those bands with the Qiaquick gel extraction kit 32 (the weight of HLA-A*0201 was 5. بينما كان اذلالم على آلة األشعة مافوق البنفسجية قمنا بتعيني حدود البقع )الصورة 14( مث نقلناه إىل وعاء عميق مث قطعنا البقع جيدا مع إزالة اذلالم الزائد )الصورة 15( وضعنا كل بقعة يف أنبوب خاص )الصورة 16( مث قمنا بأخذ وزن كل أنبوب بداخلو البقعة من اذلالم وأنبوب فارغ لنحصل على الوزن الصايف للهالم لنبدأ بتصفيتو عرب رلموعة إستخالص ىالم 32 For more detail see MEGBI training courses book part I page

55 0.30g, and for β0-m was 1.31g) then we stored the purified DNA at -20 C until use. كياكويك )الوزن الصايف للهال 0201 كان 1.32 غ وبيتا كان 1.31 غ( وبعدىا قمنا حبفظ الدنا ادلنقى على - 20 م حىت إستعمالو يف ادلراحل القادة. Figure 13: marking off the border of bands/ الاورة 13: تعيين أطراف البقع Figure 14: cutting the DNA band from the / gelالاورة 14: إزالة الهالم عن بقع الدنا ووضعهم Figure 15: DNA band in eppendorf tube / الاورة 15: وضع بقع الدنا في األنبوب تحضير الناقل / vector 4.2 Preparation of To digest the vector we assembled the following components in a microcentrifuge tube: 3 μl pet vector 3 μl 02X restriction enzyme buffer 1 µl (10 20 U) EcoR I restriction enzyme 03 μl Nuclease-free water brought to volume 32 μl Total volume We incubated at the appropriate temperature (usually 37 C) for 2 4 h in the water bath. We ran a 3 μl sample on an agarose gel to check the extent of digestion (fig.16), when digestion is complete, we added 1µl of calf intestinal alkaline phosphatase (we diluted 0.2µl of calf intestinal alkaline phosphatase in 0.8 µl of water for obtain 0.05U) directly to the remainder of the digestion. لقطع الناقل قمنا جبمع ادلكونات التالية يف أنبوب النابذة: 3 ميكرولي ت من الناقل.pET 3 ميكرولي ت من منظم أنزمي القطع ميكرولي ت ) وحدة( من أنزمي القطع إيكو.آر.وان 23 ميكرولي ت من ماء خال من النيوكياز. احلجم النهائي يف األنبوب 30 ميكرولي ت. قمنا حبضن األنبوب دلدة ساعتني على 37 م يف حوض ماء. ومن مث وضعنا 3 ميكرولي ت من العينة على ىالم األغاروز لنتحقق من قطعو )صورة 16( وعندما تأكدنا من تقطيعو أضفنا 1 ميكرولي ت من أنزمي الفوسفاتيز القلوي ادلعوي للعجل )ولكن لنحصل على تركيز 0.05 وحدة من األنزمي أضفنا 0.8 ميكرولي ت من ادلاء على 55

56 We incubated at 37 C for 30 min in the water bath then we injected the sample in 4 wells in the gel (fig.17), we ran the gel to separate the linear plasmid from nicked and supercoiled species. We visualized the DNA band with a long wave UV light source (fig.18) and we cut the band from the gel using a clean razor blade. 0.2 ميكرولي ت من األنزمي( مباشرة إلبقاء عملية القطع ووضعناه يف حوض ماء على 37 م دلدة 30 دقيقة مث قمنا بوضعو يف 4 فتحات من اذلالم األغاروزي لتنقيتو )الصورة 17( بعد ىجرة البقع على اذلالم كما يف ادلرحلة السابقة قمنا بتقطيعهم من اذلالم الزائد بواسطة مشرط حاد ونظيف )الصورة 18(. : Avoid over exposure to the light source, which can cause nicks and double strand breaks in the DNA. We balanced the bands to begin that purification with QIA quick gel extraction kit 33 Resuspended the final product in a total volume of 32 μl (usually about 52 ng/μl DNA). Assume recoveries in the range of 50% for the ligation step. Then we stored the treated vector at 20 C until use. : جيب ذبنب تعريض الزائد للهالم على األشعة ما فوق البنفسجية ألن ذلك قد يسبب شقوق وكسر للحبلني ادلزدوجني للدنا. ومن مث قمنا بقياس وزن البقع لنبدأ بعملية تنقيتهم من اذلالم بواسطة رلموعة إستخالص ىالم كياكويك. احلجم النهائي من ادلنتج ىو 30 ميكرولي ت وديكن حفظو على - 20 مº إىل حني إستعمالو مرة أخرى. Figure 16: visualization the band of the digestion vector in الاورة 16: رؤية بقع دنا / gel the الناقل المقطع على الهالم Figure 17: injection the samples in 4 الاورة :17 وضع العينات في / gel wells in 4 فتحات في الهالم Figure 18: observation the bands on UV-machine / الاورة 18: مالحظة البقع على ألة األشعة ما فوق البنفسجية 33 Refer to MEGBI training courses book part I page

57 عملية الربط / Ligation 4.3 We prepared 3 eppendorfs tubes and we marked each eppendorf tube (the first for the ligation of vector with HLA-A*0201, the second for the vector with β0-m, the third for the vector with T7 RNA Polymerase). In this step we would tried if the insert can be legated with the vector without adapter! Figure 19: Marking of each tube يف ىذه العملية قمنا بتجهيز 3 أنابيب من النابذة مع الكتابة على كل أنبوب ما الدنا الذي سيحتويو ( مثال : األنبوب األول كتبنا عليو الناقل مع اذلال 0201 الثاين الناقل مع بينا- 2 آم أما الثالث كتبنا عليو الناقل مع يت 7 بوليمرياز الرنا(. يف ىذه ادلرحلةكنا نريد أن صلرب ىل من ادلمكن ربط الدنا الزائد بالناقل من دون الوصيلة.)adapter( ال اورة 19: تعليمكل أنبوب / We added in each eppendorf tube (1.5ml): 0 μl 02X Ligase Buffer (022 mm Tris-HCl ph 7.6, 8 μl 05 mm MgCl0 0 μl 022 mm DTT 0 μl 02 mm ATP 0 μl 52 ng/μl prepared pet vector 0 μl T4 DNA ligase, diluted (with ligase dilution buffer) Weiss units/μl 0 μl Prepared target gene insert (2.0 pmol) 0 μl Nuclease-free water to volume 02 μl Total volume Then gently we mixed by stirring with a pipet tip, we incubated at 16 C to overnight. مث أضفنا يفكل أنبوب ) 1.5 مل(: 2 ميكرولي ت من منظم الليغاز ميكرولي ت منكلور ادلاغنيزيوم.25mM 2 ميكرولي ت من.100mM DTT 1 ميكرولي ت من.10Mm ATP 2 ميكرولي ت من الناقل pet احملضر.50ng/µl 1 ميكرولي ت من ليغاز الدنا يت 4 ادلخفف )مع ادلنظم ادلخفف ليغاز( وحدة/ ميكرولي ت. 2 ميكرولي ت من الدنا احملضر pmol( 0.2(. 2 ميكرولي ت من ماء خال من النيوكلياز. 20 ميكرولي ت احلجم النهائي. ومن مث مزجناىم بلطف بواسطة ادلمصة صعودا ونزوال وتركناىم على 16 م طيلة الليل. 57

58 عملية النقل / Transformation 4.4 Transformation into host strain for cloning In this step we would to prove if the ligation of the insert with the vector without adapter was possible or no, for this reason we transformed them in the host strain for cloning then we platted them and if in the next day the colonies was amplified on the plate this mean that ligation is possible and vice versa. For this step we brought a plate contains e.coli from the hospital because when we worked in the e.coli that presented in our lab we found it death and not lost the time we worked this step as follows: 1- We took some colonies from the plate then we putted it in 5ml LB medium. 2- We incubated over night at 37 C with shaking 3- In the second day we observed a big quantity of e.coli in the medium we distributed the medium in 4 eppendorf tubes and we chilled them on ice for 10 min, centrifuged them at 4000 rpm for 10 min at 4 C, discarded the supernatant and resuspended the pellets each in 600 µl of cold 0.1M calcium chloride, leaved them on ice for 25 min, centrifuged them at 4000 rpm for 10 min at 4 C, resuspended each pellet in 60 µl of cold 0.1M calcium chloride (this step for competent cells) then we let some microliter for the next step and we stored the remaining at - 20 C 4- We did the transformation as follows: عملية النقل إلى داخل خاليا اإلستنساخ يف ىذه ادلرحلة أردنا أن نقوم بتجربة ربط الدنا الزائد بالناقل من غري ادلوصل )adapter( وللتأكد من ذلك جيب نقل ادلنتج من عملية الربط إىل داخل خاليا البك تيا وزرعها على وسط غذائي مالئم مث مراقبتهم يف اليوم التايل فإذا تبني أن خاليا البكترييا تكاثرت فهذا يعين أن عملية الربط قد صلحت والعكس صحيح. وذلذه التجربة جلبنا بك تيا مزروعة من ادلستشفى وذلك ألن البك تيا اليت كانت موجودة عندنا يف ادلخترب وجدناه ميتة بينما كنا صلهزىا لنعمل هبا ولكي ال خنسر الكثري من الوقت بإنتظار إحضار بكترييا أخرى من الشركة قمنا بالعمل ببكرييا ادلستشفىكما يلي: - 1 أخذنا بعض من البك تيا ادلزروعة يف صحن ب تي ووضعناىم يف 5 مل من وسط آل يب. - 2 حضناىم طيلة الليل على 37 مº مع اإلىتزاز. - 3 يف اليوم التايل الحظنا أن خاليا تكاثرت بشكل ملحوظ يف الوسط آل يب عندئذ وزعنا كمية الوسط اليت كانت يف األنبوب إىل 4 أنابيب نابذة ووضعناىم يف الثلج دلدة 10 دقائق ومن مث نبذناىم على 4000 آر.يب.آم دلدة 10 دقائق على 4 مº مث إرمي السائل وأضف 600 ميكرولي ت من كلورايد الكالسيوم 0.1M البارد على ادل تقد يف األنبوب وإتركو يف الثلج دلدة 25 دقيقة ومن بعدىا إنبذىم على 4000 آر.يب.آم دلدة 10 دقائق على 4 مº مث أضف 60 ميكرولي ت منكلورايد الكالسيوم 0.1M على ادل تقد يف األنبوب )ىذه ادلرحلة ىي لفتح اخلاليا( وأخريا تركنا بعض ادليكرولي ت لنعمل هبم يف ادلرحلة التالية وقمنا بتخزين الباقي يف الثالجة على - 20 مº. 58

59 We pipetted 20 µl aliquots of cells e.coli from the hospital into pre-chilled tube we added 1 µl of each ligation reaction (we added the pet that without cutting as control positive), stirred gently to mix and we returned to the ice we incubated 5 min on ice, we placed the tubes in a 42 C water for exactly 30 sec. (do not shake), we placed the tubes on ice for 2 min, we added 80 µl LB medium to each tube, kept the tube on ice until all have received LB, incubated for 1 hour at 37 C with shaking. 5- We Platted them onto plate contain LB medium with ampicillin then incubated overnight at 37 C 6- We observed in the second day colonies on the pate and that mean the ligation is possible and we can continue with the expression. 7- For the plate that contains the pet (the control positive) we took some colonies from it and we putted it the 2.5ml LB medium with ampicillin then we incubated it overnight at 37 C for hours. 8- In the next day we centrifuged it and we purified it with Qiaprep spin Miniprep kit then we stored the result at -20 C. - 4 قمنا بعملية النقل كالتايل: أخذنا 20 ميكرولي ت من خاليا البك تيا ووضعناىم يف أنبوب بارد وأضفنا 1 ميكرولي ت من كل من تفاعل الربط )وأضفنا أيضا إحدى األنابيب الناقل pet لوحده كتحكم إجيايب( حركناىم بنعومة خللطهم ووضعناىم يف الثلج دلدة 5 دقائق مث يف ادلاء الساخن على 42 م دلدة 30 ثانية )من دون ربريك( مث يف الثلج مرة ثانية دلدة 2 دقيقتني وأضفنا 80 ميكرولي ت من وسط آل يب لكل أنبوب وتركناىم يف الثلج إىل أن يصل الوسط إىل اجلميع يف األنبوب مث وضعناىم يف احلاضنة دلدة ساعة على 37 م مع التحريك. - 5 مث زرعناىم على صحن ب تي احملتوي على وسط آل يب مع األمبيسيلني ومن بعدىا وتركناىم طيلة الليل على 37 م. - 6 ويف اليوم الثاين الحظنا رلموعات متكاثرة من البك تيا على الصحن وىذا يعين أن عملية الربط شلكنة وديكننا تكملة العمل يف التعبري عن الربوتني. - 7 أما بالنسبة لصحن الناقل الذي أخذناه كتحكم إجيايب أخذنا منو بعض رلموعات البك تيا ووضعناىم يف 2.5 مل من وسط آل يب مع األمبيسيلني وتركناىم طيلة الليل على 37 م دلدة ساعة. - 8 ويف اليوم التايل نبذناىم مث قمنا بتصفيتهم بواسطة رلموعة "كيابرب سبني مينيربب" والناتج الذي حصلنا عليو خزناه على - 20 م. 59

60 Figure 20: A0) take some colonies from the plate. A0) put the colonies in LB medium and let overnight at 37 C with shaking, next day observe the result as in A3, continue with the competent cells, and the transformation. الاورة 22: أ( خذ بعض مجموعات البكتريا من الاحن أ 2 ( وضعهم في وسط آل بي وتركهم طيلة الليل على 37 م مع التحريك في اليوم التالي الحظنا أن الوسط تغير لونو كما في أ 3 أكمل في فتح خاليا البكتربا ومن ثم في عملية النقل Figure 21: after preparing the LB medium with the ampicillin, B0) put it in the plate with attention from introducing air bubbles, B0) take some microliter from the transformation and plate it on the medium as fig. B3, B4) الاورة 21: بعد بحضير وسط آل بي مع اآلمبيسلين ب 2 ( ضعو في الاحن مع اإلنتباه من تكوين فقاقيع من الهواء ب 2 ( خذ بعض الميكروليتر من عينة الناقل وازرعهم على الوسط كما في ب 3 ( وب 4 (. 60

61 Figure 22: C0) after plating, incubate the plates overnight at 37 C, C0) in the next day take some colonies from the plate and put it in LB medium with ampicillin as C3) and let it between 12-16h at 37 C. ج: 1( بعد عملية الزرع أترك الاحون طيلة الليل على 37 م ج 2 ( في اليوم التالي خذ بعض المجموعات من الاحن وضعها في وسط آل بي مع أمبيسيلينكما في ج 3 ( الاورة 22 وأتركهم ما بين ساعة على 37 م. Figure 23: this the result in each step when worked in the purification of plasmid not cutting for restored at - الاورة 23: ىذه األنابيب ىي نتيجكل مرحلة من مرحلة تنقية الناقل الغير مقطع والذي سيحفظ على - 22 م. /.20 C Transformation into expression host عملية النقل إلى داخل الخاليا المعبرة We tried in this step to work in host for cloning and we added in it the T7 RNA Polymerase recombinant with the pet vector (step of ligation) and we added it with the transformation of HLA- A*0201 recombinant and β0-m recombinant then we platted them onto petri dishes and we continued the protocol until ELISA test for see if there was a complex that meaning there is a يف ىذه ادلرحلة قمنا بتجربة العمل على اخلاليا البكتريية ادلخصصة لإلستنساخ مع إضافة جني بوليمراز الرنا ت 7 ادلؤتلف مع الناقل pet )الذي جهز يف مرحلة الربط( وأضفناه معكل عملية نقل سواء كانت نقل ىال 0201 ادلؤتلف أو بيتا 2 -آم ادلؤتلف مث زرعناىم على صحون ب تي وأكملنا هبم العمل حىت فحص إيليزا لنرى إن كان ىناك مركب فهذا يعين أن ىذه ادلرحلة صحيحة والعكس صحيح. 61

62 م 4 protein and this step is correct and vice versa. To do this first should be prepared the host strain, second should be done the transformation, the plating then the extraction the protein from the strain and purified it, finally should be done the ELISA test. We took the e.coli top 10 from -70 C and that should be competent and we worked in it but when we plated onto petri dish we didn t saw in the second day any colonies, for this reason we think that the e.coli was died but for prove that we putted some micro liter of this e.coli into 5ml LB medium and we incubated them at 37 C with shaking for overnight and in the second day we saw the amplification of e.coli and that meaning the e.coli wasn t died but it wasn t competent, we did the competent for the e.coli as follows: We dispensed the 5ml of sample that in the tube in 4 eppendorf tube 1.5ml and we let them in glace for 10 min, then we centrifuged them at 4000 rpm at 4 C for 10 min, we discarded the supernatant and we added 600µl of CaCl2 0.1M cold, we let them in glace for 25 min then we centrifuged at 4000 rpm for 10 min at 4 C finally we resuspended each pellet in 60µl of CaCl2 0.1M cold, then we stored 3 eppendorf tubes at -20 C and we let one eppendorf for use it in the transformation. Secondly, we did the transformation as follows: We pipetted 20 µl aliquots of cells top 10 into prechilled tube we added 1 µl of each ligation reaction (first tube contains ligation of HLA-A0201 and T7RNA polymerase, second it contains the ligation of β0-m and T7RNA polymerase), stirred ولفعل ذلك جيب أوال ربضري سلسلة اخلاليا ثانيا جيب نقل ادلنتج من الربط إىل اخلاليا احملضرة ومن مث زرعهم ومن بعدىا إستخراج التعبري الربوتييين من سلسلة اخلاليا وتصفيتو وأخريا جيب التأكد منو بواسطة إيليزا. يف البداية أخذنا بك تيا إشرييكي القلونية top 10 من درجة - 70 م حيث جيب أن تكون مفتوحة إال أن أثناء العمل وزرعها على صحن ب تي مل يتبني لنا يف اليوم التايل أهنا تكاثرت ذلذا السبب ظننا بأهنا ميتة وللتاكد من ذلك قمنا بوضع بعض ادليكرولي تات يف 5 من وسط آل يب وتركناىم طيلة الليل على 37 م مع التحريك ويف اليوم الثاين الحظنا أن البك تيا تكاثرت وىذا يوكد بأن البك تيا ليست ميتة ولكن كانت غري مفتوحة لذا قمنا بعملية فتحها على الشكل التايل: وزعنا ال 5 مل من وسط آل يب ادلتكاثر فيها البك تيا على 4 أنابيب نابذة ) 1.5 مل( وتركناىم يف الثلج دلدة 10 دقائق مث نبذناىم على 4000 آر.يب.آم دلدة 10 دقائق على 4 م ومن بعدىا رمينا السائل الطائف يف األنبوب ووضعنا 600 ميكرولي ت من كلورايد الكالسيوم 0.1M البارد مث تركناىم يف الثلج دلدة 25 دقيقة مث نبذناىم على 4000 آر.يب.آم دلدة 10 دقائق على وأخريا رمينا السائل الطائف يف األنبوب ووضعنا 60 ميكرولي ت من كلورايد الكالسيوم 0.1M البارد مث قمنا بتخزين 3 أنابيب على - 20 م وتركنا أنبوب لنكمل العمل بو يف عملية النقل. ثانيا قمنا بعملية النقلكالتايل: أخذنا 20 ميكرولي ت من خاليا top 10 ووضعناىم يف أنبوب مربد وأضفنا 1 ميكرولي ت من تفاعل الربط ( ألا نبوب األول حيتوي على رابط ىال 0201 مع رابط بوليمراز الرنا ت 7 الثاين حيتوي على رابط بيتا 2 -آم مع رابط بوليمراز الرنا ت 7 ( حركناىم بنعومة خللطهم وأعدناىم إىل الثلج دلدة 5 دقائق مث وضعناىم يف ادلاء الساخن على 42 م دلدة 30 ثانية )من دون ربريك( مث يف الثلج مرة ثانية دلدة 2 دقيقتني وأضفنا 80 ميكرولي ت من وسط آل يب 62

63 gently to mix and we returned to the ice, incubated 5 min on ice, placed the tubes in a 42 C water for exactly 30 sec. (do not shake), turned the tubes on ice for 2 min, added 80 µl LB medium to each tube kept the tube on ice until all have received LB, incubated for 1 hour at 37 C with shaking. then we platted them onto 2 plates contain LB medium with ampicillin then incubated overnight at 37 C, we observed in the second day colonies on the plate, we took some colonies from each tube and putted in 2.5ml LB medium contains ampicillin we incubated them for hours at 37 C then we centrifuged them at 4000rpm for 5 min, resupended the pellets in µl of tris HCl (10mM) and we added in it 25 µg lyososyme, 10 µl phenylmethylsulfonylfluoride (50mg/ml), 0.08 µl DNase (1000U) 0.08 µl RNase (10mg/ml) 1 µl EDTA (1mM), incubated at 22 C for20 min, heat shock for 40s in a boiling water, centrifuged at g for 20min, washed the pellet with 250 µl 10 mm tris HCl, PH 8, dissolved it in 125 µl of 100mM tris HCl, PH8, 8M urea, then should be centrifuged at 4 C for 1 hour at g but because we didn t have a centrifuge it speed more than g we centrifuge them at g for 2 hours and saw a pellet for this reason we continued in this way, لكل أنبوب وتركناىم يف الثلج إىل أن يصل الوسط إىل اجلميع يف األنبوب مث وضعناىم يف احلاضنة دلدة ساعة على 37 م مع التحريك. مث زرعناىم على صحنني ب تي احملتوي على وسط آل يب مع األمبيسيلني ومن بعدىا تركناىم طيلة الليل على 37 م ويف اليوم التايل الحظنا تكاثر البك تيا على الوسط فأخذنا بعض من اجملموعات ادلتكاثرة ووضعناىم يف 2.5 مل من وسط آل يب مع األمبيسيلني وتركناىم على 37 م دلدة ساعة ومن مث نبذناىم على 4000 آر.يب.آم دلدة 5 دقائق ومن أضفنا على ادل تقد يف أسفل األنبوب ميكرولي ت من تريس ضبض اذليدروكلوريك )0.1M( وأضفنا عليو 25 ميكروغرام من الاليزوزمي 10 ميكرولي ت من فنيل ميثيل سولفونيل الفلورايد )50mg/ml( 0.08 ميكرولي ت من أنزمي الدنا )1000U( و 0.08 ميكرولي ت من أنزمي الرنا )10mg/ml( 1 ميكرولي ت من إي.دي.يت. يآ )1mM( مث تركناىم على 22 م دلدة 20 دقيقة ومن بعدىا قمنا بصدمة حرارية لألنبوب لدة 40 ثانية يف ماء مغلية مث نبذناىم على g دلدة 20 دقيقة ومن مث قمنا بغسل ادل تقد يف أسفل األنبوب ب 250 ميكرولي ت من تريس ضبض اذليدروكلوريك )10mM( درجة احلموضة 8 ومن مث ذوبو يف 125 ميكرولي ت من تريس ضبض اذليدروكلوريك )100mM( درجة احلموضة 8 مع اليوريا )8M( مث كان جيب نبذىم على 4 م دلدة ساعة على g ولكن دبا أن النابذة ادلوجودة يف ادلخترب السرعة القصوى لديها g قمنا بنذ األنبوبني على g دلدة 2 ساعتني وعندما الحظنا أن ىناك كمية من ادل تقد يف أسفل األنبوب أكملنا العمل على ىذا النحو 63

64 Figure 24: after incubation the colonies that contain the insert recombinant,d0) centrifuge them at 4222rpm for 5 min and obtain the result as D0), then resuspend the pellet with the buffer as D3) and incubated at 00 C for 02 min as D4) then transfer them to microcentrifuge tube, heat shock 42s as D5). الاورة 24: بعد حضن المجموعات التي تحتوي على الدنا في الوسط د 1 ( انبذ الوسط على 4222 أر. يب.آم لمدة 5 دقائق وبعدىا ستحال على النتيجةكما في الاورة د 2 ( ومن بعدىا ذوب المترقد بالمنظمكما في د 3 ( واحضنو على 22 م لمدة 22 دقيقةكما في د 4 ( وانقلهم إلى أنابيب نابذة وقم بادمة حرارية لهم على 42 ثانيةكما في د 5 (. We purified the protein of HLA-A2020 and β0-m by anion exchange chromatography Q sepharose fast flow (QFF) as follows: 1. We filled the syringe with the start buffer, removed the stopper and connected the column to the syringe with the provided connector, drop to drop to avoid introducing air into the column. 2. Removed the snap-off end at the column outlet. 3. Washed out the preservatives with 5 column volumes of start buffer, at 1ml/min for the HiTrap 1ml. 4. Washed with 5 column volumes of elution buffer. 5. Finally equilibrated with 5 column volumes of start buffer. 6. applied the sample (the protein of HLA-A0201) at 1ml/min for HiTrap 1ml using a syringe fitted to the luer connector 7. Washed with at least 5 column volumes of start buffer or until no material appears in the effluent. قمنا بتصفية بروتيني اذلال 0201 وبيتا 2 -آم بواسطة غروماتوغرافيا تبادل اإليون السيفاروزي للتدفق السريعكما يلي: 1. أمألنا اإلبرة دبنظم البدء مث أزلنا السدادة اليت على العامود وأوصلنا العامود باإلبرة بالرابط ادلناسب وحبذر ولتجنب دخول اذلواء على العامود وضعنا نقطة نقطة من اإلبرة على العامود إىل أن يلصق بو. 2. أزلنا النهاية ادلضافة عند أسفل العامود. 3.قمنا بغسل ادلواد يف العامود ب 5 مرات من حجم العامود دبنظم البدء على 1 مل/بالدقيقة لكل 1 مل من عامود "ىاي ترب". 4. قمنا بغسل ب 5 مرات من حجم العامود دبنظم اإلستخراج. 5. أخريا قمنا بتعديل العامود ب 5 مرات من حجم العامود دبنظم البدء. 6. وضعنا العينة من الربوتني ادلراد نتقيتو )اذلال 0201 (على 1 مل/بالدقيقة لكل 1 مل من عامود "ىاي ترب" بإستخدام اإلبرة ادلوصلة الرابط ادلناسب. 64

65 8. Eluted with 5-10 column volumes of elution buffer. 9. After completed elution, regenerated the column by washing with 5 column volumes of regeneration buffer (elution buffer) followed by 5-10 column volumes of start buffer. The column is now ready for a new sample. 10. Applied the sample (the protein of β0-m) at 1ml/min for HiTrap 1ml using a syringe fitted to the luer connector. 11. Washed with at least 5 column volumes of start buffer or until no material appears in the effluent. 12. Eluted with 5-10 column volumes of elution buffer. 13. When we finished the purification of the entire sample we rinse the column with water then washed with 5 column volumes 20 % ethanol at 1ml/min for the HiTrap 1ml to prevent microbial growth. Sealed the column with the supplied stoppers. And stored at 4 C to 30 C. 7. قمنا بغسل العامود ب 5 مرات من حجم العامود دبنظم البدء. مث إستخرجنا العينة ب 5 مرات من حجم العامود دبنظم 5.8 اإلستخراج. بعد إكمال عملية اإلستخراج قم بغسل العامود ب مرات من حجم العامود دبنظم اإلستخراج مث أيضا ب 5 مرات من حجم العامود دبنظم البدء. واآلن أصبح العامود جاىزا لتنقية عينة جديدة. أضفنا العينة الثانية ادلراد تنقيتها على )البيتا 2 -آم( مل/بالدقيقة لكل 1 مل من "ىاي تراب" بإستخدام اإلبرة ادلوصولة بالرابط ادلناسب. 11. قمنا بغسل العامود ب 5 مرات من حجمو دبنظم البدء. 12. قمنا بعملية اإلستخراج بإضافة 5 مرات من حجم العامود من منظم اإلستخراج. 13. عند اإلنتهاء من نتقية العينات قمنا بغسل العامود بادلاء ومن مث باأليثانول %20 على 1 مل /بالدقيقة لكل 1 مل من "ىاي تراب" دلنع منو اجلراثيم. وأخري ا قمنا خبتم العامود بالسدادات ادلزودة وادلناسبة. ووضعنا العامود على درجة حرارة بني 4 م و 30 م. 65

66 Figure 25: E0) prepare the syringe with the provider connector and fill it with the start buffer as E0), remove the stopper from the column and connect it with the syringe by drop drop as E4) to avoid introducing air to the column. ه: الاورة 1( 25 جهز اإلبرة بالرابط المناسب ومن ثم قم بملئها بمنظم البدءكما في ه 2 ( أزل السدادة من أعلى العامود وقبل بإياال اإلبرة بالعامود قم بوضع بعض النقاط على العامود لمنع دخول الهواء كما في ه 4 (. Figure 26: after the connection of syringe with the column remove the snap-off end the column as F0) wash the column with the start buffer and the elution buffer as F3) then elute the sample in a sterile eppendorf tube as F4) then when it finish the rinsing of column seal it with the supplied stopper. الاورة 26: بعد ربط اإلبرة بالعامود قم بإزالة النهاية المضافة للعامودكما في و 2 ( ثم قم بغسل العامود بمنظم البدء ثم بمنظم اإلستخراجكما في و 3 ( ثم قم بإستخراج العينة وضعها في أنبوب نابذةكما في و 4 ( وعند اإلنتهاء من إستخراج العينة قم بغسل العامود وتنظيفو ثم أغلق الفتحات بالسدادات المناسبة. Finally we began in the ELISA test as follows: In the first day we dilute 5 µl of pan specific mouse anti HLA class I antibody, W6/32 in 1ml of carbonate buffer (0.1M) PH 9.6, and we putted 50 µl/well in A1, A2, A3, A4, A5, C1, C2, C3, C4 and we let it at 4 C for overnight. In the second day we added 350 µl/well 10% w/v protein block to block the residual bind, then we washed twice with 600 µl/well of 0.05% tween in PBS at room temperature to remove the unbound W6/32 and blocking reagent, then we diluted the purified recombinant HLA-A0201 molecule 100 fold into 100µl 0.3 mm tris maleat buffer, PH 6.6, وأخريا بدأنا يف فحص إيليزاكما يلي: يف اليوم األول قمنا بتخفيف 5 ميكرولي ت من pan specific mouse anti HLA class I antibody, W6/32 يف 1 مل من منظم الكاربونات )0.1M( درجة احلموضة 9.6 ووضعنا 50 ميكرولي ت/يف كل من الفتحات التالية: أ 1 أ 2 أ 3 أ 4 أ 5 ج 1 ج 2 ج 3 ج 4 وتركناىم على 4 مº طيلة الليل. ويف اليوم الثاين أضفنا 350 ميكرولي ت/بالفتحة من %10 من الربوتيني ادلانع دلنع أي ربط آخر ومن بعدىا قمنا بغسل الفتحات مرتني ب 600 ميكرولي ت/بالفتحة من %0.05 من التويني يف PBS على درجة حرارة الغرفة إلزالة الW6/32 الغري مربتط بالفتحة وإيقاف ما تب 66

67 containing human β0-m (100nM), peptide(optimal 1nM to 1µM) and lutrol F-68 (1g/L) and we incubated them at 18 C for 48 hours. In the fourth day of ELISA test we diluted 2 µl fo the sample reaction in 10 µl of protein block with 40 µl PBS 0.05% Tween then we added 50 µl of the sample in each this well C1, C2, C3, C4 and we added on A1,A2, A3, A4, A5, the same sample but without dilution and we let the plate for 2h at 4 C, then we washed 6 times 300µl/well with PBS 0.05% Tween at room temperature, and for detecting the binding complex, we incubated the plate for 1h at 4 C, with 49 µl of post primary block + 1 µl of protein block, then we washed 6 times 300µl/well with PBS 0.05% Tween, we added one drop of Novolink polymer /well and incubated them for 30min at room temperature to enhance the detection, then we washed 6 times 300µl/well with PBS 0.05% Tween, we added 3 µl of 3,3 5, 5 tetramethylbenzidine hydrogenperoxide in A1, A2, A3, C1, C2, and we added 3 µl of DAB Chromogen in A4, A5, C3, C4, and we observed directly a change of color in A1, A2, A3, C1, C2 from incolor to blue color and after 30 min we observed that color was change to yellow and that was meaning there is a complex and AlHamdullillah we successful in the protocol and in our experiment. قي من الكواشف مث قمنا بتخفيف جزيئات اذلال ادلؤتلف ادلنقى يف 100 ميكرولي ت من منظم )0.3mM( tris maleat buffer درجة احلموضة 6.6 وحيتوي على البيتا 2 -آم )100nM( الببتيد 1µM( )1nM to واللوترول ف- 68 )1g/l( وتركناىم دلدة ساعة على 18 مº دلدة 48 ساعة. ويف اليوم الرابع من فحص إيليزا قمنا بتخفيف 2 ميكرولي ت من تفاعل العينة يف 10 ميكرولي ت من الربوتيني ادلانع مع 40 ميكرولي ت من PBS مع %0.05 من التويني مث أضفنا 50 ميكرولي ت من العينة يف كل من الفتحات التالية ج 1 ج 2 ج 3 ج 4 وأضفنا يف الفتحات أ 1 أ 2 أ 3 أ 4 أ 5 نفس زلتويات العينة ولكن من غري زبفيفها مث تركناىم على 4 مº دلدة ساعتني وبعدىا قمنا بغسل الفتحات 6 مرات ب 300 ميكرولي ت من PBS مع التووين على درجة حرارة الغرفة وإلستبان ادلركب ادلؤلف من اذلال والببتيد أضفنا 49 ميكرولي ت من بوست براديريي بلوك مع 1 ميكرولي ت من الربوتيني بلوك وتركناىم دلدة ساعة على 4 مº ومن مث قمنا بغسل الفتحات 6 مرات ب 300 ميكرولي ت من PBS مع التووين مث أضفنا نقطة من النوفولينك بوليمر على كل فتحة وتركناىم دلدة 30 دقيقة على درجة حرارة الغرفة لتحفيز اإلستبيان ومن مث قمنا بغسل 6 مرات ب 300 ميكرولي ت من PBS مع التووين ومن هبا أضفنا 3 ميكرولي ت من 5 -tetramethylbenzidine 3,3 5, hydrogenperoxide يف الفتحات التالية أ 1 أ 2 أ 3 ج 1 ج 2 وأضفنا 3 ميكرولي ت من DAB Chromogen يف الفتحات أ 4 أ 5 ج 3 ج 4 و الحظنا أن اللون يف الفتحات أ 1 أ 2 أ 3 ج 1 ج 2 قد تغري من اللون الشفاف أىل اللون األزرق وبعد 30 دقيقة الحظنا أن اللون األزرق ربول إىل اللون األصفر وىذا يعين أنو يوجد مركب وأننا احلمدهلل صلحنا يف الربوبوكول ويف ذبربتنا. 67

68 ز) Figure 27: G3) dilute the w6/30 (G0) with the carbonate buffer (G0) and put it the wells as G4. الاورة 27: ز 3 ( خفف 2( w6/32 مع منظم الكاربونات )ز 1 ( ضعهم في الفتحاتكما في ز 4 (. Figure 28: H0) put PBS with tween for the washing and discard them the waste as H0) then dry it by a towel as H3) then repeat the washing as the same method. ح: 1( ضع PBS مع التويين للغسل ثم إرميو في المهمالتكما في ح 2 ( ثم جففو على المنشفةكما في ح 3 ( ثم أعد عملية الغسل على نفس الطريقة. الاورة 28 68

69 ي) ي) Figure 29: add the protein bock then the post primary block then the Novolink polymer but after adding each reagent should be wash by PBS with tween. الاورة 29: أضف البروتين المانع ومن ثم البوست برايميري بلوك ومن ثم النوفولينك بوليمير ولكن بعد إضافةكل عامل يجب الغسل بالPBS مع التوين. Figure30: add drop of the Novolink polymer in each well as J0) then add in part of wells TMB (J0) and in the second part DAB (J3), J4) observe the wells that have TMB change it color to blue after many seconds. 2( وفي القسم الثاني DAB 3( ي 4 ( الحظ أن الفتحات الاورة 32: أضف قطرة من النوفولينك بوليمير فيكل فتحةكما في ي 1 ( ثم أضف في قسم من الفتحات الTMB التي تملك TMB تغير لونها إلى األزرق بعد ثواني Figure 31: observe the result after 30 min the color blue change to yellow in the wells A1, A2, A3, C1, C2, that have the TMB. الاورة 31: الحظ النتيجة بعد 32 دقيقة اللون األزرق تغير إلى األصفر في الفتحات التي تملك TMB أ: 1 أ 2 أ 3 ج 1 ج 2. 69